Table of Content

05 February 2012, Volume 32 Issue 2

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MiR-424 negatively regulates adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells

2012, 32(2):  114-118. 

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Object To investigate the effect of miR-424 on adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs). Methods Expression level of miR-424 during adipogenic differentiation of hAMSCs was detected by Real-Time PCR. hAMSCs were transfected with synthetic miR-424 mimics using siPORT NeoFX Transfection Agent to increase the level of miR-424 in hAMSCs, and then cultured in adipogenic differentiation medium. Oil red O staining was used to observe the accumulation of lipid droplets in hAMSCs. Expression levels of key adipogenic transcription factor PPARγ and adipogenic-related genes were detected by Real-Time PCR and Western blot. Results The expression of miR-424 decreased after hAMSCs were induced to differentiate into adipocytes. Compared to control group, the level of miR-424 was significantly increased in miR-424-mimics-transfection group. Besides，decreasing of lipid droplets accumulation was observed by Oil red O staining on Day 8 of adipogenic differentiation. Moreover, the levels of PPARγ and adipogenic-related markers were also down-regulated in the miR-424-mimics transfection group. Conclusion miR-424 could negatively regulate adipogenic differentiation of hAMSCs.

MicroRNA-373 regulates the osteogenisis of human adipose tissue-derived Flk1+mesenchymal stem cells

2012, 32(2):  119-122. 

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Objective To evaluate the effect of microRNA-373（miR-373） on osteogenic differeniation of human adipose tissue-derived Flk1+ mesenchymal stem cells (MSCs). Methods Flk1+MSCs were transfected with miR-373 mimics using Lipofectamin2000 and then induced to differentiate into osteoblasts. The osteogenic efficiency was detected by ALP staining and Alizarin red staining as well as maker genes expression through real-time PCR. Results The ALP staining and Alizarin red staining showed significant difference between two groups. Compared to control group (NC group), the expression of osteogenic maker genes RUNX2 (0.543±0.021), ALP (0.556±0.024) and OC (0.499±0.017) were significantly reduced in the mimics-transfected group (miR-373 group) (P<0.05). Conclusion miR-373 could regulate the osteogenisis of Flk1+ MSCs negatively.

Induction of mesenchymal - epithelial transition of human adipose-derived mesenchymal stem cells in vitro

2012, 32(2):  123-127. 

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Objective To explore the feasibility of the induction of mesenchymal - epithelial transition of human adipose-derived mesenchymal stem cells in vitro. Methods MSCs were isolated from human adipose and induced with activin A, RA, BMP7 in vitro. The morphological changes were observed and the expressing of protein vimentin and β-catenin were analysed by Western blot. The mRNA expression levels of vimentin, E-cadherin, ZO-1 and β-catenin were compared between induced and non-induced groups by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Results After inducing, hAD-MSCs morphology changed from spindle to orbicular-ovate, and expressed epithelial identification marker. The mRNA expression levels of E-cadherin, ZO-1 and β-catenin were significantly higher（P<0.05）, vimentin was lower in induced group than those in non-induced group（P<0.05）. Conclusion Human adipose derived mesenchymal stem cells can be induced into epithelial-like cells in vitro.

Comparison of immuno-modulatory properties between bone-marrow derived MSCs and adipose derived MSCs

2012, 32(2):  128-132. 

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Objective To compare MSCs’ immuno-modulatory property between bone-marrow derived and adipose derived MSCs. Methods Phenotype analysis of both MSCs populations was detected by flow-cytometry. To analyse immuno-modulatory effect of MSCs, proliferation, cell cycle, activation, apoptosis and Th1/Th2 balance were analyzed after co-culturing lymphocytes and MSCs.　Results Phenotype analysis showed that AMSCs had the similar expression of markers with BMSCs. Both AMSCs and BMSCs could inhibit T cells proliferation and activation. Also MSCs’ effect on Th cell differentiation into Th1 or Th2 was similar between AMSCs and BMSCs. Nevertheless, BMSCs could suppress activated T cell apoptosis, while AMSCs could not. Conclusion AMSCs and BMSCs showed similar immuno-modulatroy effect on T lymphocytes, however the difference effect on activated T cell apoptosis of the two cell populations was still unclear and the possible mechanism would be studied.

Sca-1+ bone marrow mesenchymal stem cells transplantation for the alleviation of delayed type hypersensitivity in mouse

2012, 32(2):  133-137. 

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Objective To observe the immunoregulatory effects of Sca-1+ bone marrow mesenchymal stem cells (BM-MSCs) on the delayed type hypersensitivity (DTH) mouse. Methods Balb/c mice were randomly divided into two groups, control group and MSCs infusion group. MSCs infusion group mice were intraperitoneally injected with Sca-1+ BM-MSCs from Balb/c mice on 0, 2 and 6 d, while control group mice were injected with normal saline. All the recipient mice were then injected with C57BL/6 splenocytes into the dorsal flank on 7 and 14 d into the hind footpad. After 24 h, footpad thickness, cytokines levels in the peripheral blood and the proportion of regulatory immune cells in spleen were measured by Micrometer, ELISA and FACS, respectively. Results Footpad thickness of MSCs infusion group mice (0.368 ? 0.126 mm) was significantly lower than the control group (0.731 ? 0.111 mm, p < 0.01). In the peripheral blood of MSCs infusion group mice, levels of IL-10 and TGF-β were higher than that of control group mice; while levels of IL-12 and TNF-α were lower than that of control group (p < 0.05). The proportion of regulatory T cells and dendritic cells in the MSCs infusion group mice exceeded that of control group mice (p < 0.05). Conclusion Transplantation of BM-MSCs alleviated DTH in vivo via up-regulating the levels of anti-inflammatory cytokines and regulatory immunocytes.

The protection effect and mechanism of slug on irradiation of rat intestinal epithelial cells

2012, 32(2):  138-142. 

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Objective:To investigate the protection effect and mechanism of slug overexpression on irradiation of rat intestinal epithelial cells . Methods: The study are the intestinal crypt epithelial cells (IEC6) and the IEC6 transfected with Slug cDNA in vitro.The cells are irradiated with 6 Gy60Co gamma rays.48 hours after the irradiation, Apoptosis indexes are calculated.The expression of PUMA are observed by the Immunohistochemistry and Western-blot.Result: The apoptosis indexes in IEC6 by the irradiation of 60Co gamma rays after 48 hours is significantly higher than in pcDNA3-Slug cDNA and in pcDNA3-Slug cDNA without irradiation(15.6+1.54 versus 5.1+1.7; 2.21+0.36; P<0.01). The expression of PUMA in IEC6 by the irradiation of 60Co gamma rays after 48 hours is significantly higher than in pcDNA3-Slug cDNA by Western-blot (0.79+0.12 versus 0.16+0.01; P<0.01). The Immunohistochemistry and Weatern blot assay results are consistent.Conclusions: Upregulation of Slug can restrain the upregulation expression of PUMA in irradiated cells and the apoptosis of IEC6.

Effect of Transforming Growth Factor Beta 1 on Neonatal Rat Latent Transforming Growth Factor Beta Binding Protein 2 in Cardiomyocytes

2012, 32(2):  143-148. 

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Objective: To explore transforming growth factor beta 1 (TGF-β1) induced latent transforming growth factor binding protein 2 in neonatal rat cardiomyocytes with its underlying signal transduction pathway. Methods: Primary cultured cardiomyocytes were obtained from neonatal rats by enzymatic digestion method. LTBP2 gene and its protein expression were examined by Real-time PCR, Western blot analysis and immunocytochemistry. In order to further study the signal transduction pathway of TGF-β1，inhibitors of TGF-β1 signaling pathways were used to inhibit the effect of TGF-β1 and to observe the expression change of LTBP2. Results: The stimulation effect of TGF-β1 was dose-dependent, LTBP2 gene expression began to increase after 2 ng/mL ( P < 0.05) of stimulation, peaked at 5 ng/mL (P < 0.05) and reached a plateau after 10 ng/mL (P < 0.05). The stimulation effect of TGF-β1 was also time-dependent, with the stimulation time extension, the expression of LTBP2 was increased, peaked at 12 h and declined at 24 h (P < 0.05). Immunocytochemistry showed that TGF-β1 increased LTBP2 levels. Further signaling pathway study demonstrated that TGF-β1 induces expression of LTBP2 in cardiomyocytes via ERK pathway and PI3K pathway. Conclusions: TGF-β1 up-regulated LTBP2 expression in cardiomyocytes via ERK pathway and PI3K pathway.

Suppress the expression of Fli-1 through lentivirus-based vectors mediated RNAi

2012, 32(2):  149-153. 

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【Abstract】 Objective To observe the inhibition of lentiviral vector-mediated RNA interference on the expression of Friend leukemia integration-1 gene in mouse erythroleukemia cell line（ CB3 ） and provide powerful technical support for follow-up experiments. Methods Small interfering RNA that is directed towards FLI-1 gene was designed and RNAi mediated with lentivirus-based vectors was constructed. Those colonies with positive PCR result were sequenced. After the packaging and the titer assaying, the lentivirus were used to infect the CB3 cells. Then, the expression of GFP was observed, and the inhibition of the expression of FLI-1 was analyzed via both RT-PCR and Western Blot approaches. Results The lentivirus RNAi vectors with target gene were successfully constructed and the titer of concentrated virus was 1.5E+9TU/ml.Compared to the negative and non-infected controls, the expression of FLI-1 gene was significantly inhibited after the CB3 cells were infected by lentivirus（P<0.001）. Conclusions The lentivirus RNAi vectors toward FLI-1 could be used to infect the CB3 cell line. After delivering this sort of vectors into the CB3 cells, the experession of FLI-1 was inhibited dramatically. Current research has provided a stable infection tool for CB3 cell line and this will facilitate the follow-up experiments.

Analysis cKit and BRAF mutations in 43 Chinese patients with ocular melanomas

2012, 32(2):  154-157. 

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Objective This study was designed to investigate the frequency of cKit/BRAF gene mutations in Chinese patients with ocular melanoma. Methods Forty-three ocular melanomas (30 choroidal melanomas ,13 conjunctival melanomas) were analyzed for mutations in exons 9,11,13,17,18 of cKit and in exon 15 of BRAF in genomic DNA by PCR amplification and sanger sequencing. Results In the choroidal and conjunctival melanoma subtypes, the frequency of cKit mutations was 16.7% (5/30) and (1/13), respectively. A single conjunctival melanoma harbored BRAF mutation and choroidal mealanoma lack BRAF mutation. Conclusion For the first time, this study suggests the frequency of cKit mutations was 16.7% in Chinese patients with choroidal melanomas and the cKit inhibitor might play their roles in Chinese patients with choroidal melanomas.

Effcts of electroacupuncture on mRNA and protein expression of eNOS and protein expression of MMP-9 in rats after focal cerebral ischemia/reperfusion

2012, 32(2):  158-163. 

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Abstract: Objective To investigate the effect of electroacupuncture on mRNA and protein expression of eNOS and protein expression of MMP-9 in rats after focal cerebral ischemia/reperfusion. Methods The SD rats were received filament occlusion of the right middle cerebral artery for 2 hours followed by reperfusion for 1d, 3d, 7d, respectively. Rats were randomly divided into control group, model group and EA group. After 2h of the reperfusion, EA was selected at bilateral “Hegu” point (LI 4) in the EA group. Immunohistochemical method was selected to detect the expression of eNOS protein in the cortical ischemic region. RT-PCR method was used to detect the expression of eNOS mRNA. The expression of eNOS and MMP-9 protein was detected by Western Blotting. Results Compared with the control group, the protein and mRNA expression of eNOS were increased significantly in the model and EA groups (P<0.01). Compared with the model group, the mRNA and protein expression of eNOS were increased significantly in EA groups (P<0.01). The protein expression of eNOS arrived peak in reperfusion 3d subgroups: control group (0.4291±0.0173), model group (0.7374±0.0252), EA group (0.8536±0.0212), the difference with the three groups had a statistic value (P<0.01). Compared with the control group, the protein expression of MMP-9 were increased significantly in the model and EA groups (P<0.01). The expression of MMP-9 protein was deceased significantly in the 1d subgroup in EA (P<0.01), but increased significantly in the 3d, 7d subgroups (P<0.01). Conclusions EA treatment up-regulates the protein and mRNA expression of eNOS and the protein expression of MMP-9 in rats after focal cerebral ischemia/reperfusion.

Effects of Hypoxia on migration and invasion of esophageal carcinoma Eca109 cells in vitro

2012, 32(2):  164-169. 

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Objective To explore the effect of hypoxia on migration and invasion of hypoxia-inducing reagent to mimic tumor hypoxic microenvironment. mRNA and protein levels of HIF-1α, E-cadherin and MMP-2 at different hypoxic culture phases were detected by semi-quantitative RT-PCR and immunohistochemistrym, respectively. After being treated with rapamycin combined with hypoxia, the protein expression of HIF-1α, E-cadherin and MMP-2 were assayed by western blot. The effect of rapamycin combined with hypoxia on migration and invasion were tested by cell scratch assay and transwell chambers. Results Under hypoxia, mRNA level of HIF-1α had no significant change, while its protein level increased obviously; mRNA and protein levels of E-cadherin were down-regulated, but the mRNA and protein levels of MMP-2 were up- regulated. Under hypoxia, rapamycin inhibited the expression of HIF-1αand MMP-2, but E-cadherin was up-regulated. The migration and the number of invading cells decreased (P<0.05) after the treatment of rapamycin under hypoxia. Conclusion Hypoxia can increase protein level of HIF-1α in esophageal carcinoma, which promote migration and invasion possibly through down-regulating E-cadherin and up-regulating MMP-2 .

Effects of panax notoginseng saponins on reducing damage and inhibiting apoptosis in rabbits with lung ischemia reperfusion injury

2012, 32(2):  170-174. 

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AIM: To explore relationship between apoptosis in the lung tissues and lung ischemia reperfusion injury, and observe effects of PNS by regulating apoptosis in lung ischemia reperfusion injury. METHODS: Single lung in situ ischemia reperfusion animal model was used. Eighty-four Japanese white rabbits were randomly divided into control group (Control), ischemia reperfusion 1h group (IR1h), IR3h, IR5h, Panax Notoginseng Saponins 1h group (PNS1h), PNS3h and PNS5h. The terminal deoxynucleotidyl transferase-mediate dUTP end labeling (TUNEL) and in situ hybridization were used to observe apoptosis; Fas、FasL mRNA and Caspase-3 mRNA expression in various phases of lung ischemia reperfusion; DNA fragmentation was observed by polyacrylamide gel electrophoresis (PAGE). RESULTS: Cell apoptosis (IR5h:22.08±1.93;Control:2.04±0.67) in lung tissues of rabbits with lung ischemia reperfusion were significantly higher than in Control group (all of P<0.01), especially in pulmonary vascular endothelial cells and alveolar epithelial cells. Fas/FasL mRNA and Caspase-3 mRNA (IR5h:0.241±0.029; Control:0.121±0.015) were up-regulated in lung tissues of lung ischemia reperfusion injury than in those of Control groups (all of P<0.01). The PNS suppressed apoptosis as well as expression of Fas/FasL mRNA and Caspase-3 mRNA (PNS5h:0.199±0.020, IR5h:0.241±0.029, all of P<0.01). There was a significant correlation between expression of Fas/FasL mRNA、Caspase-3 mRNA and cell apoptosis (all of P<0.01). DNA ladder appeared in IR5h group, but not seen in PNS5h group. CONCLUSIONS: The protective effects of PNS include suppressing the activation of Fas/FasL system and blocking apoptosis in lung tissues of lung ischemia reperfusion rabbits.

Mechanism of PLCepsilon gene blockage on invasive power of human bladder cancer cells

Li-Ping OU Chun-Li LUO

2012, 32(2):  175-180. 

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Objective The aim of this study was to construct a shRNA expression plasmid against gene PLCε and to observe the inhibition of PLCε gene expression in bladder cancer cell line T24.Methods pGenesil-PLCε plasmids were constructed and insert T24 cells,then RT-PCR, Western blot analysis was taken to know about inhibition of PLCε gene expression after the transfection of plasmids. invasive power of T24 were measured before and after transfection by the membrane invasion culture system(Transwell chamber), gelatin enzymography and immunochemistry of cells. Results It was confirmed by digesting and sequencing that the two recombinant plasmids had been constructed successfully. The inhibition rate of PLCε mRNA was 76.0% and 73.9%， and this of protein expression was 65.4% and 64.8% . The invasion number of T24 transfected with P1(25.8±6.2) and P2(26.8±5.8) are both lower than the the group of HK-A (33.8±5.7 )and group without transfection(34.8±6.9) (P<0.01); MMP-2,MMP-9 had lower expression in gelatin enzymography assay and imunocytochemistry of cells than the groups HK-A and control(p<0.01). Conclusion:RNAi of PLCε might decrease invasive power of bladder cancer cells so as to inhibit the development of tumor.

CD4+CD25+FoxP3+regulatory T cells suppress specific cellular immune responses in active pulmonary TB patients

2012, 32(2):  181-185. 

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Objective To identify whether CD4+CD25+FoxP3+regulatory T cells(Treg) are expanded in patients with active pulmonary tuberculosis(TB) and to characterize Treg functions in the modulation of antigen-specific responses. Methods The frequency and the immune function of circulating regulatory T cells were compared in the patients with active pulmonary TB and heathy control subjects using flow cytometry analysis,proliferation assays and determination of cytokines. Results The percentage of CD4+CD25+FoxP3+regulatory T cells within the total CD4 CD4+ population was significantly increased in the peripheral blood in active pulmonary TB patients than that in heathy control subjects(p<0.01).The peripheral blood mononuclear cells(PBMCs) of pulmonary TB patient had significantly higher cellular proliferation and IFN-γ production in response to both Bacille Calmette Guerin(BCG) and early secretory antigenic target-6(ESAT-6) compared to PBMCs from healthy donors(all p<0.05).In active pulmonary TB patients, the peripheral blood CD4- T cells had higher BCG-induced IFN-γ and IL-10 production (1289.62±519.01 and 1045.40±534.12, respectively) compared to PBMCs(624.50±261.13 and 377.00±249.56, respectively)(all p<0.05). These CD4+CD25+FoxP3+regulatory T lymphocytes are capable of suppressing IFN-γ and IL-10 production in response to both BCG and ESAT-6 in peripheral blood CD4+CD25- cell from TB patients. Conclusions CD4+CD25+FoxP3+ Regulatory T cells expanded in patients with active pulmonary TB may therefore contribute to the pathogensis of human TB by suppressing specific Mycobacterium tuberculosis cellular immune responses.

Effects of 9-cis-retinoic acid on cell cycle and expressions of CyclinD1 and CDK4 in thyroid squamous cell carcinoma cell line SW579

2012, 32(2):  186-190. 

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Abstract：Objective To study the effects of 9-cis retinoic acid(9-cisRA) on cell cycle and expressions of CyclinD1 and CDK44 in thyroid squamous cell carcinoma cells(SW579).Methods Treated with different dose of 9-cisRA (final concentration of 10-7 mol/L、10-6 mol/L、5×10-6mol/L、10-5mol/L). the proliferation of the cells was observed with MTT and the cell cycle was measured by flow cytometry;the CDK4 and CyclinD1 mRNA expression levels were determined by RT-PCR;CDK4 and CyclinD1 protein expression were determined by western blotting. Results After cells were treated with 9-cisRA the proliferation index decreased markedly with a dose-dependent manner.Flow cytometry analysis showed the percent of G1 phase cells was significantly higher than that of the control groups and the percent of S phase cells was lower than that of the control groups .The cell cycle was arrested at G1 stage;The mRNA expression of the CDK4 was not increased; the mRNA expression of CyclinD1 was. descreased dramatically; Distinct changes in the expressions of CDK4 protein was not observed; the expressions of CyclinD1 protein was. descreased dramatically; Conclusions 9-cisRA inhibited the proliferation of SW579 cell in a dose-dependent manner,It was related with down-regulation of CyclinD1 and arresting sw579cell at G1 phase.

Primary research and identification of associated proteins in serum of cervical cancer

2012, 32(2):  191-194. 

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Primary research and identification of associated proteins in serum of cervical cancer Rong Chun-hong, Shen Keng, Yang Jia-xin, .Department of Obstetrics and Gynecology Peking Union Medical College Hospital,Beijing,100730,China [Abstract] Objective: To discover different proteins between cervical cancer and control group , then we separate, purify and enrich the interest proteins and identify them by MS. Methods We use SELDI-TOF-MS to analyze the difference peptide profiling and proteins between serum of cervical cancer patients and the control group, interested proteins are subsequently separated and enriched by ion chromatographic fractionation, SDS-PAGE ,at last the proteins are identified by LC-MS/MS analysis, SELDI-TOF-MS is used to verify the existence of interest proteins. Results 12 different proteins are identified in serum between cervical cancer and the control group(P<0.05). We separate, purify and identify interest proteins whose M/Z is 15960Da and get 8 kinds of proteins according the database. The most adjacent to 15960Da molecular weight comes from transthyretin (TTR), which there are some subjects showed it’s correlation to tumor. The TIF1-β protein, namely KAP-1/KRAB/TRIM28, arose us by it’s biologic action, because it’s expression can inhibit or activate tumor related genes then promote the development of malignant tumor. Conclusions We are the first in China who applying the technology course that discover different proteins by SELDI-TOF-MS , purify and enrich interested proteins monitoring by SELDI-TOF-MS, identify it by LC-ESI-MS/MS. We purify and identify the protein whose M/Z is 15960Da, data of IPI gives us 8 kinds of possible proteins, in which the molecule weight of TTR is the most close, but TIF-1β has the most important biologic behavior . [Key words] cervical cancer SELDI-TOF-MS TIF1-β TTR

TThe relationships between hippocampal NMDA receptor and the configuration of NOS positive neurons in myenteric nerve plexus in stomach in CUMS in rats

2012, 32(2):  195-200. 

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Objective Investigate the involvement of hippocampal glutamate and NMDA receptor in the effect of CUMS on gastric function and enteric nervous system (ENS). Methods Unpredictability chronic mild stress (CUMS)-induced depression model was established in SD rats, intra-hippocampal microinjections of Glu and non-competitive antagonist of N-methyl-D-aspartic acid (NMDA) receptor (MK-801) were respectively adopted by rat brain stereotaxic coordinates. The behavioral observations were conducted by sucrose preference test and forced swimming test. The intra-gastric pressure were recorded and the expression of Nitric oxide synthase (NOS) positive neurons in myenteric nerve plexus in stomach were detected as well. Results CUMS rats showed depression-like behavioral changes and weaker gastric motility compared with control. Pretreated with MK-801 could reverse these CUMS effect, while microinjection of Glu, instead of CUMS, could increase the duration of immobility in forced swimming test, but has no effect on gastric motility. CUMS rats had lower expression of NOS positive neurons(73.74±16.38/LPF, P<0.05) and positive ganglions number(4.25±1.34/LPF, P<0.05) compared with control, but the number of NOS positive neurons in every ganglion was increased(6.55±2.37, P<0.05). Pretreated with MK-801 could rescue the decline of the number of positive ganglions. Conclusions CUMS could regulate the activity of NMDA receptor in hippocampus, and then change the configuration of NOS positive neurons in ENS, weaken the gastric motility. While the effects of CUMS on depression-like behavioral changes due to both Glu and NMDA receptor in hippocampus.

Study of CDH1 gene germ-line mutation and promoter methylation status in hereditary diffuse gastric cancer

2012, 32(2):  201-206. 

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Abstract：0bjective To detect the expression of CDH1, screen the germ-line mutation of CDH1 exons and evaluate CDH1 promoter methylation status in a family with hereditary diffuse gastric cancer(HDGC) in China．Methods Fifteen members of a family with HDGC were visited, peripheral blood samples and tumor specimens were collected. The expression of CDH1 gene was detected by　Immunohistochemistry and Western blot. By PCR and direct sequencing germ-line mutation of 16 CDH1 exons were screened. PCR and Clone Sequencing were used to investigate the status of CDH1 promoter methylation. Results In proband and another gastric cancer patient(number 2 member), the protein expression of CDH1 were reduced in mucosa near the tumor, and lost in the tumor. In 11 members(including proband), a single nucleotide substitution of C?T (SNP) in exon 14(mRNA 2377 locus) was found. No germ-line mutation of 16 exons was detected in all members. Compare with normal mucosa, hypermethylation was found in the tumor and mucosa near the tumor of proband and number 2 member. Conclusions In this family with HDGC, abnormal expression of CDH1 may be a cause of gastric cancer. But germ-line mutation of 16 exons was not the cause of CDH1 gene inactivation in the tumor, CDH1 promoter hypermethylation may be one of reasons inducing inactivation of the gene.

Clinical Significance and Dynamic Change of Plasma Levels of sCD14 in Patients with Sepsis

2012, 32(2):  207-210. 

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Objective: To explore the soluble cluster of differentiation antigen 14(sCD14) dynamic changes and clinical significance on patients with sepsis. Methods: 60 patients were diagnosed with sepsis according to the diagnosis criterions and selected as sepsis group from the emergency intensive care unit of Chaoyang Hospital affiliated Capital Medical University during the period from August 2009 to March 2010. 20 healthy persons were chosen as control group. Sepsis group was divided into multiple organ dysfunction syndrome (MODS) and non-MODS two subgroups according to MODS clinical diagnosis criterions. sCD14 concentration was detected and APACHEⅡscore were calculated on 1st , 3rd and 7th days after the patients were diagnosed sepsis in sepsis group. Results: Compared with control group, sCD14 concentration was obviously higher in sepsis group（P<0.01）. Compared with non-MODS subgroup, MODS subgroup has more higher sCD14 concentration [（5.3±2.4）vs （3.8±2.8）μg/mL P<0.05 ]and APACHEⅡscores( P<0.05). sCD14 concentration and APACHEⅡ scores had correlation( P<0.05). Conclusions: sCD14 concentration was high on patients with sepsis. MODS patients had high sCD14 concentration and APACHEⅡ scores, patients condition were more critical.

Exogenous EGFP labeling affects Tumor Cells Biological Behavior

2012, 32(2):  211-216. 

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Abstract: Objective To study the influence of exogenous EGFP labeling on biological behavior of tumor Cells in vitro . Methods Using liposome transfection and lentivirus infection respectively, exogenous EGFP carried by different vectors was expressed in different tumor cell lines. Stable exogenous EGFP expressing cell lines were selected for further analysis. The growth was measured by MTT assay. The distribution of cell size was tested by Cell Counter（CasyTT）. The invasion was measured by Boyden-chamber assay. Results Stably-expressing of exogenous EGFP human colon cancer cell line HCT116-GFP, HCT116-EGFP, COLO 320DM-EGFP and murine dendritic cell sarcoma cell line DG6-EGFP was successfully established. After expression of exogenous EGFP, the growth of HCT116-GFP cells was similar to that of HCT116 cells, while the growth of HCT116-EGFP cells, COLO320DM- EGFP cells and DG6-EGFPcells was significantly decreased. After expression of exogenous EGFP, the cell volume of HCT116-EGFP cells was bigger than that that of HCT116 cells, the cell volume of DG6-EGFP cells was similar to that of DG6 cells. After expression of exogenous EGFP, the invasion of HCT116-EGFPcells was significantly lower than that of HCT116 cells, the invasion of DG6-EGFP cells was similar to that of DG6 cells. Conclusion Exogenous EGFP labeling of tumor cells may or may not affect the biological characteristics of the tumor cells in vitro, indicating that before the use of these models, researchers should evaluate its effects on the relevant parameters.

RNA interference gene silencing Bax mitochondrial pathway of apoptosis of chondrocytes

2012, 32(2):  217-221. 

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[Abstract] Objective Using cultured rat chondrocytes to detect the influence of the expression of silence gene Bax interfered with RNA to cell apoptosis via mitochondria. Methods SD rat cartilage cells were isolated and cultured in vitro; expression of silence gene Bax was interfered with Bax siRNA;. expression levels of mRNA and proteins were detected by RT-PCR and Western blot; cells activity were tested by MTT assay; detect apoptosis rate was detected by Annexin V-FITC/PI double labeling assay; expression levels of Bcl-2, Cytochrome C proteins were detected by Western blot. Results after interfering with Bax siRNA for 24h, Bax mRNA and protein expression were significantly reduced. Apoptosis was inhibited, and cell survival rate increased. Moreover, in cells which silenced at Bax gene, Cytochrome C protein level decreased, while expression of Bcl-2 protein levels elevated. Conclusion To interfere silence gene Bax with RNA could inhibit apoptosis of rat chondrocytes and promote their survival, which may be associated with the mitochondrial pathway.

The Liver Transplantation in patients with previous extrahepatic malignancies

2012, 32(2):  222-225. 

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Objective To study the indication of liver transplantation for the patients with previous extrahepatic malignancies. Methods 2 patients with hepatocelleuer carcinoma or liver cirrhosis,had suffered with rectal cancer and breast cancer, both of them had been receive radical surgery before, receiving orthotopic liver transplantation and postoperative treatment of individualize Immunosuppressive therapy, liver cancer patients after conventional chemotherapy. Results The patients recovered well after surgery, followed up for 79 months and 34 months, with good liver function, no tumor recurrence. Conclusion Previous extrahepatic malignancy should not be considered a contraindication for liver transplantation, selection of suitable patient and to take individual immunosuppressive and chemotherapy, can achieve satisfactory results.

Construction of Adenovirus expressin vector and Gain Gene Silencing Effect in Suspension Cells

2012, 32(2):  226-232. 

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Abstrac: Objective Constructed recombinant adenovirus to efficiently obtain gene silencing in suspension cell. Methods Inserted 76 nt DNA stem-loops into plasmid pRNAT-H1.1/Adeno, in which an incorporated GFP reporter allows convenient tracing of all steps in viral production. In this pilot study, placental growth factor (PLGF) was targeted by this vector. The recombinant shuttle vector were linearized with PmeⅠ and treated with alkaline phosphatase, then cotransformed with pAdeasy-1 into BJ5183 cells by electroporation. Positive clones were selected and confirmed by PacⅠdigestion. Before transfected the packaging cell line HEK 293, the recombinant adenovirus plasmid must be cut by PacⅠ. After three times amplified in HEK293 cells, high titter viral particles were harvested and quantified by OD260 optical absorbance and TCID50 method individually. Finally, the viral particles were utilized to transfect two suspension cells (NCI-H 250 and NCI-H 209, the human small cell lung cancer cells). And using real-time PCR to test the target gene expression and Transwell cancer cell migrate test to analysis cancer cells’ function. Results Monitoring GFP expression and using real-time PCR method, efficient and specific silencing of PLGF gene was found in NCI-H 250 and NCI-H 209 cells after adenovirus transfection 48 h. And a significant reduction of migration was observed in target gene silenced cancer cells. Conclusion Our results indicate a prospective application of this shRNA expressing recombinant adenovirus system in those cells transfected difficultly by other methods.

New Strategies in Tetracycline-regulatable System

2012, 32(2):  233-236. 

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Preclinical studies have confirmed the efficacy of viral vectors as a gene delivery tool，and recent clinical trials have shown promising results with this strategy. But before the clinical application，viral gene vectors should be reconstructed to make it can be regulated in a time- and dose-dependend fashion. Usually，a gene regulation system will be inserted into the vector genome. The tetracycline regulatory system (Tet system) is by far the most frequently used in gene therapy. By adjusting tetracycline or its derivatives (such as doxycyline) in medium or in vivo，the expression of target gene will be switched on or off. The development of Tet system and its application is discussed in this review.

Research progress on erectile dysfunction following brachytherapy for prostate cancer

Jian CHEN

2012, 32(2):  237-240. 

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