Objective To observe the replicative senescence of rat articular chondrocyte cultured in vitro so as to provide reference for the succeeding experiment of using medicine interfere and reverse the cataplasia of tissue engineering cartilage or probing cataplasia mechanism．Methods Different generations(P1, P2, P3 and P4) of the chondrocytes were detected with the methods of histochemistry for β-galactosidase (β-gal)， electronmicroscope for ultromicrostructure， immunocytochemistry for proliferating cell nuclear antigen (PCNA)，alcian blue stain for content and structure of sulfatglycosaminoglycan (GAG) of extracellular matrix (ECM)，reverse transcriptionpolymerase chain reaction (RTPCR) for content of collagen Ⅱ，flow cytometry for cell life cycle and proliferative index(PI) to observe senescence of chondrocytes．Results In the 4th passage，the chondrocytes emerging quantitively positive express of β-gal，cyto-architecture cataplasia such as caryoplasm ratio increasing and karyopycnosis emerging under electronmicroscope ，cell life cycle being detented on G1 phase(83．8％)，while in P1, P2, P3 the content of G1 phase was 79.1%, 79.2%, 80.8% respectively. In the 4th passage, PI decreased(16．2％)，while in P1, P2, P3, it was 20.9%, 20.8%, 19.2%. The positive percentage of PCNA，the content of GAG(long chain molecule) and the positive expression of collagen Ⅱ diminished,all detections above were significantly different (Plt;0.01) when compared the 4th passage with the preceding passages．Conclusion Chondrocytes show the onset of senescence in the 4th passage．
ObjectiveTo summarize the recent research progress on pathogenesis of human arrest defective 1(ARD1) protein in colorectal cancer and treatment process. MethodsSearched the related literatures from the databases such as CNKI, PubMed and so on, the relevant ARD1 in the development, diagnosis and treatment of colorectal cancer were reviewed. ResultsARD1 has effect of anti colorectal cancer, it can inhibit the proliferation and promote apoptosis of colorectal cancer cells, and improve the sensitivity of colorectal cancer cells to anticancer drugs at the cellular level. The treatment is mainly through the induction of cancer cell apoptosis or (and) decreased the proliferation ability of cancer cells, thus delaying the disease process. However, it is still in the research stage of animal experiments, which can not be directly applied to clinical practice. Conciusions ARDl study on the mechanism of anti colorectal cancer cells has become the focus of research with animal research and promotion, and provide new therapy concepts and measures for diagnosis and treatment of colorectal cancer.
OBJECTIVE: To investigate the effects of dexamethasone on the proliferation and differentiation of bone marrow stromal cells(MSC). METHODS: MSC were isolated and cultured in vitro. After treatment with different concentrations of dexamethasone (0, 10-10, 10-9, 10-8, 10-7 and 10-6 mol/L), the proliferation and alkaline phosphatase (ALP) activity of MSC were measured to evaluate the effect of dexamethasone on the biological characteristics of MSC. RESULTS: Dexamethasone inhibited cell proliferation. With the increase of concentration of dexamethasone, the effect was enhanced, which was more significant when the concentration of dexamethasone was over 10-8 mol/L. At the same time, dexamethasone promoted the activity of ALP. This effect was enhanced with the increase of concentration of dexamethasone, but the alteration was small when the concentration of dexamethasone was over 10-8 mol/L. The effects increased with the time. The activity of ALP was enhanced 2 to 4 times with the dexamethasone for 6 days. CONCLUSION: Dexamethasone inhabit the proliferation of MSC, while induce them to differentiate into osteoblasts. The appropriate concentration of dexamethasone was 10-8 mol/L.
ObjectiveTo explore the effects of exogenous estrogen receptor β1 (ERβ1) gene on the expression of human telomerase reverse transcriptase (hTERT) as well as the changes of proliferation ability in MDA-MB-231 cell line by transfecting recombinant eukaryotic expressing vector containing ERβ1 cDNA into human breast cancer MDA-MB-231 cell. MethodsRecombinant eukaryotic expressing vector containing ERβ1 cDNA was transfected into human breast cancer MDA-MB-231 cell by using cationic liposome as transfecting agent (acted as pcDNA3.1ERβ1 transfection group), empty vector group and non-transfection group acted as controls. The expression levels in both the mRNA and protein of both the ERβ1 and hTERT were tested by real-time PCR and Western blot, respectively. The change of proliferation ability in MDA-MB-231 cell was displayed by cell growth curve, and the change of cell apoptosis was detected by flow cytometry. ResultsThe expression level of ERβ1 mRNA in the pcDNA3.1-ERβ1 transfection group (0.449±0.077) significantly increased as compared with the nontransfection group (0.153±0.035) or the empty vector group (0.160±0.020), P=0.001 or P=0.000. The expression level of ERβ1 protein in the pcDNA3.1-ERβ1 transfection group (0.847±0.065) significantly increased as compared with the non-transfection group (0.356±0.050) or the empty vector group (0.390±0.030), P=0.001 or P=0.000. The expression level of hTERT mRNA in the pcDNA3.1-ERβ1 transfection group (0.127±0.020) significantly decreased as compared with the non-transfection group (0.283±0.025) or the empty vector group (0.283±0.049), P=0.001 or P=0.002. The expression level of hTERT protein in the pcDNA3.1-ERβ1 transfection group (0.147±0.023) significantly decreased as compared with the non-transfection group (0.783±0.025) or the empty vector group (0.802±0.019), P=0.001 or P=0.002. The rate of cell apoptosis in the pcDNA3.1-ERβ1 transfection group 〔(6.15±0.94)%〕 was higher than that in the non-transfection group 〔(1.41±0.42)%〕， P=0.001. Cell proliferation curve showed that proliferation ability significantly decreased in the pcDNA3.1-ERβ1 transfected groups as compared with the non-transfection group (Plt;0.05). ConclusionERβ1 could inhibit cell growth of human breast cancer MDA-MB-231 cell by down-regulating the expression of hTERT.
Objective To study the effect of two cytokines, basic fibroblast growth factor(bFGF) and insulin-like growth factor-I(IGF-I), on cell proliferation in chondrocytes of adult rabbits. Methods The primary chondrocytes of adult rabbits were harvested and cultured with bFGF and IGF-I at different concentrations,respectively, as well as with the mixture of the two cytokines; the quantity of cultured chondrocytes was detected by MTT assay at the 24th, 48th and 72th hours; and the final fold increase of different groups was measured by cell count for the 3rd passage; and the proliferation index of the groups was recorded by flowing cytometer on the 14th day. Results ① The cultured chondrocytes with either bFGF, IGF-I or their mixture were significantly more than that of control group at the 24th, 48th and 72th hours (P＜0.01). ② After the 3rd passage, the final folds of proliferation were significantly higher in the groups with cytokinesthan in the control group (P＜0.01); and the final fold with the mixture ofcytokines was significantly higher than that of both IGF-I and bFGF (P＜0.01). ③ Theproliferation index was significantly higher in the groups with cytokines than in the control group (P＜0.01); the proliferation index with the mixture of cytokines was significantly higher than that of both IGF-I and bFGF (P＜0.05); besides, proliferation index was higher when cytokine was applied twice than once (P＜0.05). Conclusion bFGF and IGF-I could promote chondrocytes proliferation of adult rabbits obviously and they are synergistic in cell proliferation.
Objective To review the research progress of mesenchymal stem cells (MSCs) in burn wound repair. Methods The recent literature about MSCs involved in burn wound repair and mechanism was extensively reviewed and analyzed. Results MSCs have the capacity of self-renew, rapid proliferation, differentiation and paracrine, and promote burn wound repair through differentiating into a series of skin wound cells and regulating wound microenvironment. Conclusion MSCs have great potentials in the burn field. However, the cell survival and outcome are also facing challenges from poor microenvironment of the burn wound.
ObjectiveTo investigate effect of small interfering RNA (siRNA) targeting inhibition of growth hormone receptor (GHR) on proliferation and invasion of human liver cancer line SMMC-7721. MethodsSMMC-7721 cells were transfected with siRNA targeting human GHR by GenMuteTM transfection regent.The cells were divided into three groups:blank control group (non-transfected siRNA),negative control group (transfected with non-specific siRNA),and specificity transfected group (transfected with expression specifically interfere by GHR siRNA).the relative expression of GHR mRNA was detected by using real-time PCR.the expression of GHR protein was detected by using Western blot.The cell proliferation was assessed by CCK-8 assay.And the ability of invasion was examined by Transwell assay. ResultsThe expressions of GHR mRNA and GHR protein in the specificity transfected group were significantly lower than those in the blank control group (P<0.05) and the negative control group (P<0.05).Compared with the blank control group and the negative control group,the absorbance value and the number of migrating cells of SMMC-7721 cells were decreased obviously (P<0.05) in the specificity transfected group. ConclusionsiRNA targeting human GHR could reduce capability of proliferation migration and invasion of SMMC-7721 cells.
Objective To observe the proliferation and migration of endothelial cells after 30% total burn surface area (TBSA) of deep partial thickness scald, and the effect of basic fibroblast growth factor (bFGF) on angiogenesis during wound healing.Methods A total of 133 male Wistar ratswere divided randomly into normal control (n=7), injured control group (n=42), bFGF group (n=42) andanti-c-fos group (n=42). The apoptosis expression of fibroblasts was determinedwith in situ hybridization and the changes of proliferation cell nuclear antigen(PCNA), focal adhesion rinase(FAK), c-fos and extracellular signalregulated kinase(ERK) proteins expression were detected with immunohistochemistry staining technique after 3 hours, 6 hours, 1 day, 3 days, 7 days, 14 days and 21 days of scald.Results In injured control group and bFGF group, theproliferation rate of the vascular endothelial had evident changes 7 days and14 days after scald； the expression of FAK was increased 14 days after scald. ERK proteins expression was different between injury control group and bFGF group at initial stage after scald. Stimulation of ERKs by bFGF led to up-regulation of c-fos and b expression of FAK. Conclusion Exogenous bFGF extended the influence on wound healing process by ERK signaling pathway, affecting migration cascade of vascular endothelial cell. The oncogene proteins play an important role on accelerating angiogenesis duringwound healing.
ObjectiveTo investigate the influence of EZH2 gene down-regulation by RNA interference on the proliferation and invasion of human glioma cell line U251. MethodsThe recombinant plasmid of small hairpin RNA targeting EZH2 gene was constructed, and transfected into gioma U251 cells by electroporation. The expression of EZH2 mRNA and protein in the cells was detected by using reverse transcriptase-polymerase chain reaction and Western blot respectively; the viability of cells was determined by using methyl thiazol tetrazo1ium assay; and the invasiveness of U251 cells was tested by Transwell cabin. ResultsThe expression levels of EZH2 mRNA in U251 cells were detected in a significantly lower proportion in the EZH2-shRNA group (0.19±0.02) than that in the untransfected group (1.13±0.05) and the control-shRNA-GFP group (1.15±0.05). The expression levels of EZH2 protein in U251 cells were detected in a significantly lower proportion in the EZH2-shRNA group (0.20±0.02) than that in the untransfected group (1.03±0.03) and the control-shRNA-GFP group (0.97±0.06). The proliferation rates in EZH2-shRNA group were significantly decreased as compared with those in the untransfected group and control-shRNA-GFP group (24 hours after transfection:60.13%±3.15%, 100.00%±9.31%, 100.03%±9.35%; 48 hours after transfection:53.01%±3.14%, 100.00%±9.13%, 99.58%±9.27%; P<0.05) and Transwell cabin suggested that the invasiveness of U251 cells was significantly decreased (46.00±2.82, 60.67±5.71, 61.00±2.48; P<0.01). ConclusionEZH2-targeted RNA interference can reduce the proliferation and invasion of human glioma cells, which suggests that EZH2 shRNA may be a potential gene therapeutic target of human glioma.
ObjectiveTo investigate the effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), a hypoxia-inducible factor-1α (HIF-1α) inhibitor, on hypoxia induced rat pulmonary arterial adventitial fibroblasts (AFs) proliferation and collagen synthesis, and explore the molecular mechanism.MethodsUnder hypoxic condition, rat AFs were cultured in DMEM medium supplemented with 10% fetal bovine serum in vitro. The cells were divided into five groups, ie. a normoxia group, a hypoxia group and three hypoxia+YC-1 groups (treated with YC-1 at concentration of 0.01, 0.05 and 0.1 mmol/L, respectively). The cells proliferation was determined by MTT method. Collagen synthesis of AFs was measured by 3H-proline incorporation assay. The expression of HIF-1α in AFs in different conditions was measured by Western blot, and the mRNA expression of transforming growth factor-β1 (TGF-β1) was measured by reverse-transcription polymerase chain reaction.ResultsThe proliferation rate and the incorporation data of 3H-proline in the hypoxia group were significantly increased as compared with those in the control group (both P<0.01). YC-1 significantly reduced the proliferation rate and incorporation data of3H-proline induced by hypoxia in a dose-dependent manner. YC-1 could also down-regulate the expressions of HIF-1α and TGF-β1 mRNA significantly (both P<0.01). Compared with the hypoxia group, the expressions of HIF-1α and TGF-β1 mRNA decreased respectively by 65% and 61% in the hypoxia+YC-1 (0.1 mmol/L) group (bothP<0.01).ConclusionsYC-1 can inhibit hypoxia-induced AFs proliferation and collagen synthesis in a dose-dependent manner. The mechanism may relate to YC-1’s inhibitory effect on expressions of HIF-1α and TGF-β1 mRNA.