Table of Content

05 June 2014, Volume 34 Issue 6

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Expressions of FGFR2 isoforms and the relevant alternative splicing factors in human embryonic stem cells and adult stem cells

2014, 34(6):  723-728. 

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Objective To compare the expressions of FGFR2 isoforms and the relevant alternative splicing factors in human embryonic stem cells (hESCs) and their differentiated embryoid bodies (EBs), and human adult stem cells (ASCs) derived from different tissue sources, including bone marrow derived mesenchymal stem cells (BMSCs), adipose derived mesenchymal stem cell (ADSCs) and hair follicle stem cells (HFSCs). Methods The characteristics of hESCs, EBs and ASCs were confirmed by RT-PCR, immunofluorescence staining and flow cytometry analysis. The expressions of FGFR2 isoforms and the relevant splicing factors were quantified by real-time PCR. Results FGFR2 IIIc is the dominant isoform in all tested cells. Upon differentiation, the expressions of FGFR2 isoforms, as well as the IIIb/IIIc ratio in EBs were significantly increased (P<0.05). The expression of splicing factors that inhibited IIIc expression (FOX2) and inhibited IIIb expression (HnRNPA1) in EBs was increased after differentiation for 7 days and 14 days respectively (P<0.05). The expressions of FGFR2 isoforms in hESCs and HFSCs were significantly higher than in BMSCs and ADSCs, and significantly higher in BMSCs than in ADSCs (p<0.05). Expressions of multiple FGFR2 relevant splicing factors were significantly higher in hESCs and HFSCs than in BMSCs and ADSCs. Conclusion The expression profiles of FGFR2 isoforms and the relevant splicing factors in stem cells at different developmental stages and from different tissue sources are different.

Effect of Curcumin on RECK expression and methylation in nasopharyngeal carcinoma cells CNE-1

2014, 34(6):  729-733. 

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Objective To investigate the effect of curcumin on reversion-inducing-cysteine-rich protein with kazal motifs (RECK) gene expression and methylation, and then analyzed the possible mechanism. Methods nasopharyngeal carcinoma cell line CNE-1 was cultured in vitro, and stimulated by 1, 10 and 30 μmol/L curcumin for 48h, expression of RECK and DNA methyltransferases 1 (DNMT1) gene mRNA and protein were detected by Western blot and real-time PCR, respectively. RECK methylation was determined by HPLC chromatographic and mass spectrometric methods. Cell proliferation was assessed by MTT assay, and the DNA-binding activity of nuclear factor Sp1 was detected by EMSA. Results The expression level of RECK was very low in unstimulated cells, and 1~30μmol/L could decrease the protein and mRNA level (P<0.05). 30μmol/L of curcumin could decrease the promoter methylation level to 31% of the basal level (P<0.01), and the global DNA methylation level and the methylation activity of the nuclear extract were also decreased about 39% (P<0.01) and 71.6% (P<0.01), respectively. Western blot showed that curcumin could also decrease the protein and mRNA level of DNMT1 (P<0.05), and survival rate (P<0.05). In addition, the DNA-binding activity of Sp1 was decreased after curcumin treatment, as demonstrated by EMSA. Conclusion Curcumin inhibits CNE-1 cells growth by up regulation of RECK gene expression and down regulation of the its methylation.

The analysis of coding genes expression during the differentiation of neural stem cells

2014, 34(6):  734-739. 

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Objective Investigation of the functional genes expression patterns during the differentiation of neural stem cells (NSCs) by microarray assay and bioinformatics analysis. Methods Cell culture and differentiation of NSCs, cellular sorting system based on the combination of cell surface markers, bioinformatics analysis were employed to display the change patterns of functional gene pathways in astrocytes and neurons compared with NSCs. Results The results show that the multiple genes expression activities in Wnt, insulin and P53 pathways are dynamically changed in differentiated astrocytes and neurons from NSCs. Conclusion The complex interactions of functional gene pathways are important for the precise differentiation of NSCs.

Lactic Acid Regulates Phenotype Polarization and Function of Macrophages in Tumor Microenvironment

2014, 34(6):  740-745. 

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Objective To investigate that lactic acid regulates polarization of phenotype and function of macrophages in tumor microenvironment. Methods Balb/c mice were inoculated by 4T1 breast tumor cells with the mammary gland injection.The tumor tissues were harvested at different time points and detected the lactic acid concentration. The expression of M1 and M2 markers in RAW264.7 cells treated with different concentration of lactic acid were detected by RT-PCR ,FACS and Western Blot. The mouse cytokine array chip was used to evaluate the release of cytokines from RAW cells treated with 15mmol/L lactic acid. Phagocytosis and antigen presenting function was evaluated by using FACS. Results lactic acid concentration of mouse breast tumor was higher than that of normal breast tissue（P<0.05）. M2 markers of RAW cells were up-regulated and M1 markers were down-regulated by 15mmol/L lactic acid（P<0.05）.M1 cytokines secretion of RAW cells treated with 15mmol/L lactic acid was down-regulated（P<0.05）. Phosphorylated NFκB P65 was down-regulated by 15mmol/L lactic acid（P<0.05）.The phagocytosis was not affected by lactic acid, but the expression of the antigen presenting and co-stimulatory molecules were down-regulated in RAW cells treated with 15mmol/L lactic acid（P<0.05）. Conclusion lactic acid induces the M2 phenotype shift and inhibits the antigen presenting function of RAW cells. NFκB P50/P65 are involved in the phenotype polarization of macrophages.This study suggested that lactic acid plays a indispensable role in macrophages polarization in tumor microenvironment.

The establishment of genetically CDR3δ2-modified γδ T lymphocytes and its functional characterization

2014, 34(6):  746-752. 

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Objective To explore the anti-tumor effect of genetically CDR3δ2- modified γδ T lymphocytes which were generated by co-transfection of the full-length γ9 mRNA and δ2 mRNA with tumor-reactive CDR3δ into anti-CD3 antibody stimulated human peripheral blood mononuclear cells (PBMC). Methods By PCR, specific δ2 sequences containing tumor-reactive CDR3δ2 (OT3 and OT10) and γ9 chain were amplified to make recombinant plasmids pGEM4Z / δ2 (OT3) / A64, pGEM4Z / δ2 (OT10) / A64 and pGEM4Z/γ9/A64. Linearized plasmids were used as templates for the synthesis of δ2 (OT3), δ2 (OT10) and γ9 mRNAs in vitro. Synthesized δ2 (OT3) mRNA and δ2 (OT10) mRNA were co-transfected with γ9mRNA respectively into anti-CD3 antibody stimulated human PBMC. Flow cytometry and sorting were performed to isolate γ9δ2 (OT3) T cells and γ9δ2 (T10) T cells. MTT assay and ELISA were applied to detect the anti-tumor effect of these CDR3δ2 genetically modified γδ T lymphocytes. Results CDR3δ2 genetically modified γ9δ2 (OT3) T cells and γ9δ2 (T10) T cells secreted high levels of IFN-γ and TNF-α and displayed significant cytotoxicity to a variety of tumor cells. Conclusion The generation of genetically modified tumor-reactive γ9δ2 T cells is an efficient approach to enhance the anti-tumor activity of lymphocytes, which provides a novel strategy for adoptive immunity against tumors.

Promoter hypermethylation of GATA5 and its impact on clinical outcome in invasive breast cancer

2014, 34(6):  753-756. 

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Objective To detect promoter methylation status and mRNA expression level of GATA5 and to explore the relationship between methylation and mRNA expression in invasive breast cancers (IBC). Methods Promoter methylation analysis of GATA5 was performed by methylation-specific PCR (MSP) in IBC and matched normal tissues (MNT) from 62 patients. We examined expression profile of GATA5 using quantitative real-time PCR (qRT-PCR) analysis in 48 of 62 patients. Results Methylation of GATA5 was only present in IBC (48.4% (30 of 62)) (P < 0.001). mRNA expression of GATA5 in IBC showed the lower level than that in MNT (P < 0.001). For GATA5, methylated IBC only showed significantly lower expression values compared to MNT (P < 0.001). Tumors with methylated GATA5 showed significantly less fold change of GATA5 than tumors without methylation (P < 0.05). Conclusions Methylation of GATA5 maybe is one important mechanism of the gene transcriptional inactivation, and is associated with the formation and development of IBC.

Function of twist1 during neural progenitor cells differentiation in mouse cortex

2014, 34(6):  757-761. 

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Objective To screen the expression pattern of Twist1 during the development of cerebral cortex at different stages, and investigate the potential function of Twist1 in neural progenitor cell differentiation; Methods Expression pattern of Twist1 in mice developing cerebral cortex was clarified by in situ hybridization, and in utero electroporation of overexpression plasmid into the cerebral cortex neural progenitor cells was conducted to test the in vivo function of Twist1; Results Twist1 is expressed in cerebral cortex through differentiation stages tested, and showed a higher expression level in the ventricular zone, overexpression of Twist1 result in decreased cells in ventricular zone and more cells in intermediate zone(p<0.05); Conclusions Twist1 is expressed during the process of neural differentiation with a higher expression level in neural progenitor cells, and could promote neural differentiation in some extent.

KLF9 adenoviral vector construction and it’s function in anti-ROS

2014, 34(6):  762-766. 

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Objective To construct the adenoviral overexpressed vector of Krüppel-like factor 9(KLF9) and to explore the role of KLF9 in the regulation of anti-ROS genes expression. Methods The KLF9 gene is first cloned into the shuttle vector pAdTrack-CMV. The resultant plasmid is linearized by digesting with restriction endonuclease Pme I, and subsequently cotransformed into E. coli. BJ5183 competent cells with pAdEasy-1.Recombinants are selected for kanamycin resistance and recombination confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmid is transfected into 293A cells. Recombinant adenoviruses are typically generated within 7 to 12 days. The mouse primary hepatocytes are isolated from the C57BL/6J mouse and subsequently H2O2 treatment is conducted. We aslo performed KLF9 overexpression by using Ad-virus system (Ad-KLF9) in mouse primary hepatocytes isolated from.Quantitative real-time PCR (qRT-PCR) analysis was further performed to determine the expression of anti-ROS genes including catalase(CAT), manganese superoxide dismutase(SOD2) and genes were upregualted in C57BL/6J mouse primary hepatocytes following H2O2 treatment (P<0.05).The results of qRT-PCR and western blot replied that adenoviral vector of KLF9 was successfully produced with 90% infection efficiency. The expression of anti-ROS genes were also upregualted in C57BL/6J mouse primary hepatocytes after infecting with Ad-KLF9(P<0.05).Conclusion KLF9 represents its impact on the expression of anti-ROS genes.

Determination of volume change of LPS-stimulated mouse peritoneal macrophages by flow cytometry

2014, 34(6):  767-770. 

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Objective To determine the volume change of mouse peritoneal macrophages after LPS-stimulation. Methods After stimulation with LPS for 3 and 24 hours, the cell morphological changes were observed under phase-contrast microscope, the content of IL-6 in the cell supernatant was measured with ELISA (enzyme linked immunosorbent assay), and the volume changes were analyzed with flow cytometry, respectively. Results In the normal condition, mouse peritoneal macrophages are round with smooth edge and without vacuoles. After stimulated by LPS, the surface area of mouse peritoneal macrophages become larger than before. Pseudopodium and vacuoles can also be seen and as stimulation time extended, their number increased. Especially after 24 hours, mouse peritoneal macrophages turned into fusiform, and their pseudopodia fused with each other. After LPS stimulation, we found the content of IL-6 in the cell supernatant increased dramatically, illustrating the secretory pathway of inflammatory factor has been activated by LPS and the peritoneal macrophages have been activated. Flow cytometry observed increasing FSC (demonstrating cell size) by 11% (P < 0.05) after 3 hours and 20.2% (P < 0.01) after 24 hours, as well as SSC (demonstrating paticles in cell surface) increased by 21.6% (P < 0.05) after 3 hours and 68% (P < 0.01) after 24 hours. Conclusions Flow cytometry can determine the volume changes of mouse peritoneal macrophages quantitatively, providing new method for studying the activation and function of innate immunity cells.

AZD8055 inhibits proliferation and induces apoptosis in huh7 cells hepatocellular carcinoma

2014, 34(6):  771-775. 

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Objective This study is aim to investigate effects of AZD8055 on proliferation and colony formation of hepatocellular carcinoma cells. Methods The huh7 cells were divided into two groups: control group (DMSO) and AZD8055 group. MTT assay and colony formation assay were performed to detect effects of AZD8055 on proliferation and colony formation of hepatocellular carcinoma cells, respectively. Apoptosis rate was assessed by performing AnexinV/FITC double staining. Western blot was performed to detect AMPK and p-AMPK expression. Results AZD8055 inhibit proliferation of huh7 cells in a time- and concentration-dependent manner (P<0.05). AZD8055 treatment reduced colony numbers of huh7 cells and induced apoptosis compared to those untreated cells (P<0.05). AZD8055 treatment increased p-AMPK expression but didn’t change AMPK expression. Blocking AMPK activation by using AMPK inhibitor Dorsomorphin partially reversed proliferation inhibition induced by AZD8055. Conclusion AZD8055 inhibit proliferation and colony formation of huh7 cells through AMPK signaling pathway.

Knockdown of PCBP2 with small interfering RNA regulates P53 related genes and cell proliferation in glioma cells

Wei HAN Bin YIN Xiao-zhong PENG

2014, 34(6):  776-781. 

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Objective To study the effect of poly(C) binding protein 2 (PCBP2) silencing on P53 related genes and glioma cell proliferation. Methods PCBP2 gene-specific siRNA or control siRNA was transfected into three glioma cell lines (T98G, U87MG and U251). The expression levels of PCBP2 protein, P53 and its target genes PUMA and BAX, P53 co-regulator FHL2 and another member of the Four-and-a-half-LIM domain (FHL) family FHL1 were detected by Western blot. The interactions between PCBP2 and FHL family members’ mRNA were analyzed by Biotin pull-down. Cell proliferation was determined by BrdU incorporation. The apoptosis ratio of cells was measured by Hoechst staining. Results After transfection of PCBP2 siRNA, PCBP2 protein was evidently silenced; the protein levels of P53 and its target genes PUMA and BAX were increased (P < 0.05); the protein level of FHL2 was decreased (P < 0.05) but its mRNA 3’UTR didn’t bind to PCBP2, the BrdU positive cells decreased (P < 0.05); the number of Hoechst positive cells increased (P < 0.05). Conclusions Knockdown of PCBP2 can enhance the activity of P53 pathway in glioma cells with the weakened ability of cell proliferation and induction of cell apoptosis.

Establishment of an electroporation method for transfection of mRNA into anti-CD3 antibody stimulated human PBMC

2014, 34(6):  782-786. 

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Objective To establish an electroporation method to transfect mRNA into human PBMCs for the preparation of CDR3-grafted γδ T lymphocytes. Methods linearized plasmids pGEM4Z/EGFP/A64 containing EGFP cDNA fragment by the digestion with restriction endonuclease SpeⅠ were used as templates for EGFP mRNA transcription in vitro. EGFP mRNA was transfected into anti-CD3 antibody-stimulated human PBMC by electroporation instrument under different conditions with 500V and 500?s, 400V and 500?s or 300V and 500?s, respectively. The levels of EGFP expression in the cells electroporated with EGFP mRNA were evaluated through inverted fluorescence microscope and flow cytometry. The survival rates of transfected-cells were measured by trypan blue staining. Results Agarose electrophoresis showed that pGEM4Z/EGFP/A64 plasmids were well linearized by SpeⅠ digestion and EGFP mRNAs were successfully transcribed in vitro. 48 hours after mRNA-transfection, EGFP expression in cells transfected EGFP mRNAs under the condition of 500V and 500?s attained 77% which were significantly higher than those in cells transfected EGFP mRNAs under other conditions. The survival rate of transfected cells under the condition of 500V and 500?s reached up to 85%. Conclusions The electroporation platform of mRNA transfection into human PBMC with parameters of 500V and 500?s is successfully established and could be useful to prepare genetically modified T lymphocytes by mRNA transfection.

Suprachiasmatic nucleus lesion induced abnormal rhythm of circadian inflammatory pain

2014, 34(6):  787-791. 

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Objective To investigate the regulation of suprachiasmatic nucleus (SCN) on the inflammatory pain in mice at behavioral level. Methods Wheel running of circadian behavioral monitor system, the surgery of SCN lesion and the formalin induced nociceptive responses test were employed to analyze if the synchronize and/or maintain circadian function of SCN could modulate the rhythm of inflammatory pain induced by formalin in mice. Results Our results showed that the formalin induced inflammatory nociceptive responses were shown a circadian oscillation pattern in resting phase (ZT4) and active phase (ZT20), and after SCN lesion, the mice were shown an overturned pattern of nociceptive responses between ZT4 and ZT20 compared with normal control. Conclusions The circadian synchronized function is an important regulator for maintaining normal daily fluctuation patterns of inflammatory pain induced by formalin.

Effect of different loading exercise on morphology and metabolism of articular cartilage in ovariectomized female rats

2014, 34(6):  792-796. 

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Objective The aim of the present study is to investigate the influence of loading exercise on morphology and metabolism of articular cartilage in ovariectomized rats .Methods Forty female Sprague-Dawley rats were randomly assigned to 4 groups: ovariectomized (OVX), ovariectomized plus low loading exercise (low loading OVX-RUN), ovariectomized plus medium loading exercise (medium OVX-RUN), ovariectomized plus high loading exercise (high loading OVX-RUN); After 12 weeks, the following variables were compared among the 4groups. Knee joint and blood samples were collected，Test of rat serum related material were carried on the articular cartilage morphology (HE dyeing, sarranine O dyeing , toluidine) examination and immunohistochemical staining to detect the content of type Ⅱ collagen in articular cartilage. Result Accroding to the morphology and serum test results ,medium OVX-RUN group was helpful to maintain the steady state of articular cartilage (p<0.05). compared with OVX、low loading OVX-RUN group、medium OVX-RUN group, high loading OVX-RUN group was not conducive to the growth of the articular cartilage,induce thedegenerative-like changes in articular cartilage(p<0.05).Conclusion The different loading exercise has different effect on the cartilage of rats, moderate loading exercise on the prevention and treatment of osteoporosis has useful clinical significance.

High throughput mutation screening based on metal-enhanced fluorescence

2014, 34(6):  797-801. 

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Objective To develop a highly sensitive and selective method for mutation detection utilizing metal enhanced fluorescence (MEF). Methods Strand-displacement was initiated by introducing the target ssDNA and resulting in the unfolding of the capture probe. A signal probe coupled with Carboxy fluorescein(FAM) was further hybridizing with the unfolding capture probe and generating fluorescent signal. Results Comparing to normal method, the MEF design could discriminate complementary target from targets with mismatch, and holds noticeable specificity and highly sensitivity (~13 fold signal amplification with 100nmol/L target) with the detection limit of 0.1nmol/L in silver-coated 96-well plate (control group is 5nmol/L). Conclusion The MEF assay provided an alternative screening method for mutation detection.

Application of mRNA conserved sequence from Tissue-specific factors to the identification of the tissue origin of cultured cells

2014, 34(6):  802-809. 

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Objective To establish an effective and economic fast method that could identify the tissue origin of cultured cells , classify fresh derived human tissue cells and different tissue cells. Methods According to references and NCBI database, we designed 17 pairs of tissue-specific primers targeted to the conserved sequences of mRNA coding human tissue-specific factors.The mRNA isolated from the known cells was reverse transcribed to cDNA which was further amplified with these primers by PCR. Agarose gel analysis of the PCR products was employed to testify specificity and sensitivity.And the selected primers were applied to identify double-blinded unknown samples. Results Among the 17 pairs of tissue-specific primers, 8 pairs corresponding to ALB, AFP, SYN, CDH16, SFTPB, LCA, FLT, MUL2 could identify whether the cell originates from the liver, kidney, nerve, lung, hematopoietic or endothelial tissue. The prostate specific antigen (PSA) can be amplified from cells of all tissue origin. Conclusion This RT-PCR assay of tissue-specific mRNA conserved fragments provides a simple ,rapid, sensitive ,and cost-effective new method to identify cell tissue types and detect cell origin.

Clinical outcome analysis of gefitinib integrated chemotherapy in metastatic or recurrent nonsmall cell lung cancer patients

2014, 34(6):  810-813. 

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Objective To analyze the clinical outcomes between gefitinib integrated chemotherapy and chemotherapy alone among metastatic or recurrent nonsmall cell lung cancer (NSCLC) patients who failed in previous treatment with gefitinib and chemotherapy. Methods A total of 33 pairs patients with metastatic or recurrent NSCLC who had progressed on prior therapies and received gefitinib plus chemotherapy or chemotherapy alone between January 2006 and June 2011, were matched according to gender (male vs female), age(<65 vs≥65years old), Eastern Cooperative Oncology Group (ECOG) performance status (PS) (0-1 vs 2), progress free survival (PFS) of gefitinib treatment(4-6 vs >6months),number of previous chemotherapy regime and status of tumor at diagnosis, by using a matched-pair case-control study design. Results With 14.5 months of median follow-up, the overall response rates and disease control rates in the integrated-treated and chemotherapy alone groups were 9.1% versus 6.5% and 39.4% versus 30.3%, respectively. There was no statistically significant difference noted with regard to overall survival (OS) (from first gefitinib therapy to death, median, 10.4 vs 7.9 months) and PFS (median, 4.2 vs 3.3 months) between the integrated-treated and chemotherapy alone groups. Conclusions This retrospective analysis shows that there is no difference in clinical outcomes between integrated treatment and chemotherapy alone in patients with metastatic or recurrent NSCLC who failed in previous treatment with gefitinib and chemotherapy.

Proteomics analysis of antagonistic effect of Cinobutacini injection on MDR of HCC cells

2014, 34(6):  814-818. 

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Objective To investigate the mechanism of antagonistic effect of Cinobutacini injection（CINO） on multidrug resistance (MDR) of human hepatocellular carcinoma in protein expression.Methods Human hepatocellular carcinoma 5-FU resistant cell line were cultured in vitro.The cytotoxicity and the antagonistic effect of Cinobutacini injection on MDR was detected by MTT. The differential expression protein between Bel-7402/FU cells and Bel-7402/FU cells treated with 0.26μg/L CINO were detected by 2-DE and MS . The differential expression of protein FAK,CRT and TMOD3 were detected by westernblot to confirm the results of 2-DE and MS .Results The 50% inhibitory concentration(IC50) as to Bel-7402/FU cells was 0.66μg/L。0.08、0.16、0.32μg/L CINO reversed the MDR by1.56、2.18、2.99 folds respectively . there are 13 protein spots down -regulated and 1 protein spot up-regulated in Bel-7402/FU cells treated with CINO，westernblot comfirmed that portein FAK， CRT and TMOD3 were down-regulated in Bel-7402/FU cells by CINO，they were in accordance with the result of 2-DE and MS . Conclusion CINO can effect the expression of proteins related to MDR ,so as to reverse the MDR of human HCC cells.

The natural antisense transcript, Lmo4as, negatively regulated the same locus cortical coding gene Lmo4

2014, 34(6):  819-823. 

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Objective Investigation of the cis regulatory functional role of natural antisense transcript (NAT) in expression of same genome locus coding gene, Lmo4. Methods Rapid Amplification of cDNA Ends (RACE), RT-PCR, real time qRT-PCR, in situ and Luciferase reporter assay were employed to capture the full length of the NAT, to display the spatio-temporal patterns of coding gene and noncoding RNA in the development of embryonic brain, and to analyze the regulatory role of NAT in the expression of Lmo4, respectively. Results The results show that Lmo4as is a noncoding RNA transcript with a poly-A tail, and the full length sequence of this NAT transcript were cloned. Moreover, it was shown that Lmo4as function as a negative regulator on the expression of Lmo4 at the post-transcriptional level, similar with a microRNA functional role, miR-124. Conclusion The NAT Lmo4as function as a cis repressor on the coding gene Lmo4 at post-transcriptional level.

Changes of lymphocyte subsets in peripheral blood with hay fever patients in different period and its significance

2014, 34(6):  824-827. 

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Objective To investigate the change of peripheral blood lymphocytes of hay fever and its relationship with different stage of pathogenesis. Methods The peripheral blood cell counts and lymphocyte subsets of 20 cases hay fever patients in onset and non-onset period were determined by hematology analyzer and flow cytometry. Results The level of eosinophils, basophils, CD8+CD28+T cells and CD8+CD38+T cells was significantly higher (P <0.05 ), NK cells was lower (P <0.05) , and B lymphocytes, T lymphocytes, CD4+T cells, CD8+T cells and CD4+CD28+T cells had no significant change in peripheral blood of hay fever in onset period compared with those in non-onset period. Conclusions Hay fever patients’CD8+CD28+ and CD8+CD38+T cell might be in a state of immune activation in onset period, play an important role in allergic inflammation.

Pericyte mediated angiogenesis in spinal cord injury mice

2014, 34(6):  828-833. 

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Objective The aim of our study was to investigate the influence of pericytes on microvascular angiogenesis after spinal cord injury (SCI). Methods C57BL/6 mice were randomly divided into four groups: sham group, 2 days (S2), 7 days (S7) and 14 days after SCI group (S14) (n=9 per group). The injury group received a moderate impacted spinal cord injury. Perfused blood vessels were detected by FITC-LEA intravenous injection; microvessel area density (MVA) and microvessel density (MVD) were analyzed by immunofluorescence and immunohistochemistry, respectively; pericyte coverage was further calculated by immunofluorescence. Hypoxic condition (95% N2 and 5% CO2) was applied to mimic the phathological situation of SCI, endothelial tubular formation was detected by matrigel system. Results The area of perfused blood vessels, MVA and MVD at S2 were significantly decreased compared with that at sham (P < 0.001). MVA and MVD at S7 was notably increased compared with that at S2 (P < 0.05). The area of perfused blood vessels, MVA and MVD at S14 showed a markedly increase compared with that at S2 (P < 0.05, P < 0.01), which were still lower than that at sham. Pericyte coverage was decreased at S2 and S7 in mice compared with sham control. Co-culture of pericytes and endothelial cells (ECs) could markedly increase endothelia tube length compared with ECs alone under hypoxia (P < 0.01). Conclusion Pericytes might promote angiogenesis in spinal cord injury C57BL/6 mice.

Profiling of microRNAs bound to PCBP2 using RIP-Chip

2014, 34(6):  834-839. 

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Objective To screen all the microRNAs (miRNAs) bound to poly(C) binding protein 2 (PCBP2) in normal human astrocytes (HA) and glioma cells (T98G and U87MG) using ribonucleoprotein immunoprecipitation - microarray profiling (RIP-Chip). Methods The expression levels of PCBP2 protein were detected in HA, T98G and U87MG cells by Western blot analysis with the PCBP2 antibody purified from rabbit serum. With normal rabbit IgG as negative controls, the protein and RNA samples from RIP assay were enriched in the three cell lines. The protein samples were detected enrichment effect by Western blot; The puried RNAs were quantified by the NanoDrop ND-2100 and the RNA integrity was assessed using Agilent 2100.The labeled RNAs were hybridized onto the Affymetrix miRNA 3.0 microarray. The enriched miRNAs were then identified through fold change as well as P value calculated using t-test. The threshold set for up - regulated genes was a fold change > 4 and a P value < 0.05. Results RIP-certified anti-PCBP2 antibody was validated for use in ribonucleoprotein (RNP) immunoprecipitation (RIP) in conjunction with the RIP-Assay Kit for microRNA. Its ability to immunoprecipitate RNP complex and miRNAs was confirmed. 103 mature miRNAs, 1 precursor miRNA (pre-miRNA) and 1 small nucleolar RNA (snoRNA) were selected to interact with PCBP2; 15 of which exist the PCBP2 binding sites. Conclusions PCBP2 can bind to mature miRNA, pre-miRNA or snoRNA in vivo not only through the target sequence recognition but also by the formation of ribonucleoprotein complexes.

PET/CT is applied in diagnosis of Crohn’s disease

2014, 34(6):  840-843. 

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Objective Crohn’s disease (CD) is a chronic inflammatory disease involving the gastrointestinal tract with a tendency toward remission and relapse, and sometimes hardly be diagnosed until taken operation. It is said that 18F-PET/CT is a novel non-invasive technique for differential diagnosis and assessment of Crohn’s disease. Methods 3 patients were included from June 2012 to Augest 2012 in PUMCH, all have done the 18F-PET/CT. Results Patient 1, there were many abnormal areas with high FDG uptake in intestinal tract which standardized uptake value (SUVmax) were similar to in liver. Patient 2, the SUVmax was twice higher than in liver. Patient 3, it was more than 4-fold. Patient 1 and 2 were considered as CD, and patient 3 was T-cell lymphoma. Conclusions 18F-PET/CT is a novel non-invasive technique, but high-tech and expensive, for patient who is suspected for CD .

Twenty flavour aloes pil increased rats lung tissue PKC - α expression in hypoxic pulmonary arterial hypertension

2014, 34(6):  844-846. 

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SiRNA-mediated silencing of the HBx gene inhibits cell migration in human hepatocellular carcinoma cell HepG2.2.15

2014, 34(6):  847-848. 

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Effect of rosiglitazone on metastasis of BIU-87 bladder cancer cell line

2014, 34(6):  849-850. 

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Hydrogen decreases Inflammatory factors release of post-hypoxia/reaeration Injury in human lung epithelial cell line

2014, 34(6):  851-852. 

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The research advances of toll-like receptors and the atopic dermatitis

2014, 34(6):  853-856. 

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Toll-like receptor (TLR) is one kind of the most thoroughly studied pattern recognition receptors (PRRs). TLRs signaling pathways are triggered after recognization of specific ligands. TLRs trigger innate immune responses, prime antigen-specific adaptive immunity and play an important role in the allergy, antivirus activity and chronic inflammation. Atopic dermatitis (AD) is a common skin disease, characterized by severe itching, skin barrier damage, and increased susceptibility of pathogens. Here we describe the recent advances about expressions of TLRs and genetic polymorphisms in AD patients, regulation to TLRs by vitamin D in AD delopment and the role in pathogenesis of AD.

Research progress on sacral nerve stimulation treatment of bowel dysfunction after spinal cord injury

2014, 34(6):  857-860. 

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Sacral nerve stimulation as a new treatment of bowel dysfunction has been gradually applied to the clinical practice. The mechanism may be related to the regulation of afferent reflex and local reflex pathway. Stimulation parameters set individually. The main clinic evaluation indicator is Cleveland clinical constipation score and life quality score.

Molecules involved in protection of retinal pigment epithelial cells against oxidative stress

2014, 34(6):  861-865. 

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Chronic oxidative stress is involved in development of many retinal degenerative-related diseases. Oxidative stress could induce the damages of proteins, lipids, DNA and organelles in retinal pigment epithelial (RPE), and cause RPE cellular dysfunction. So far, the molecular mechanism and regulatory process of oxidative stress in RPE still remain unclear. Recent studies found that some of molecular chaperones, transcription factors and microRNAs play a critical role in protection against oxidative stress in RPE, which may provide new therapeutic strategies for retinal degenerative-related diseases.

RUNX3 and digestive system carcinoma

2014, 34(6):  866-869. 

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RUNX3(hunan Runt-related transcription factor 3) playing important roles in regulation on cell proliferation,cell proliferation and cell apoptosis is a member of the Runt-related transcription factor family.The abnormal expression of RUNX3,such as heterozygous deficiency,hyper-methylation,has significant relationship to the occurrence,development,and prognosis of malignant tumor,and especially plays important roles in the occurrence and development of gastric cancer,colorectal cancer, and liver cancer.

Historical view of PUMC: philosophy and pathway of elite education of medicine

2014, 34(6):  870-872. 

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The history of medical education at Peking Union Medical College (PUMC) is reviewed for her philosophy and learning pathway. Elite education is the core value of school performance and this supports PUMC to implement education and training and to set up a model of teaching, training and resources mobilization. The key elements of school structure, teaching principle and education strategy are discuss.