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To study the effect of the Tangshenping containing serum on the proliferation of the high glucose-induced epithelial cells of renal tubules.To prepare durg-contained serum from rats to enter into in vitor reaction system,the cellscultured via 10％ FBS-RPMI 1640 were randomly divided into 7groups:the normal group,themodel group,the Y27632 group,theirbesartangroup,the small dose Tangshenping group,the medium dose Tangshenping group and the high dose Tangshenping group and The cells were cultured in 3000 cells/well and grown in 96-well plates.Each group had 8 wells,then detect the effects of all serum sections on the proliferation of high glucose-induced epithelial cells of kidney tubules by the MTT colorimetric method after cultured for 12 h,24 h,48 h,and 60 h.Based on the results above,cell protein were extracted from each group at 24 h,and the expression of RhoA,ROCK1,α-SMA and E-cadherin in each group were detected by Western blotting.After high glucose stimulation,the shape of cell was shuttle-like or irregular triangle,the way it grew was radial;after the intervention of the corresponding serum,the shape of the cell was fiat and irregular polygonal.Started with 12h,compared with the normal group,OD value of other groups increased;at the 24h、48hand 60h,compared with the normal group,OD value of high glucose groupincreased significantly (P＜0.01);compared with the high glucose group,OD value of treatment groups decreased (P＜0.05);and 48 h,compared with the Y27632group,irbesartan groupand Tangshenping high dose group,OD value of Tangshenping low and medium dose groups decreased (P＜0.05);60 h,compared with Y27632 group,OD value of Tangshenping medium dose groups decreased;compared with irbesartan group,OD value of o Tangshenpinggroupsdecreased (P＜0.05);compared with Tangshenping high dose group,OD value of Tangshenpinglow groupsdecreased (P＜0.05) Western blotting analysis showed that compared with normal group,the expression of E-Cadherin protein in high glucose group reduced,and the expression of RhoA,ROCK1 and α-SMA protein increased;compared with high glucose group,the expression of E-Cadherin protein in each treating group increased,and the Tangshenping large dose group wassignificantly different (P＜0.01);the expression of RhoA,ROCK1 and or-SMA protein reduced,Tangshenping,the large dose group was significantlydifferent (P＜0.01);Compared with the Y27632 group,the expression of E-cadherin,ROCK1 and α-SMA protein in Tangshenping large dose group had no significant difference,while the expression of RhoAproteinreduced (P ＜0.01).Compared with theirbesartan group,the expression of E-cadherin,RhoA,ROCK1 and α-SMA protein in Tangshenping large dose group had no significant difference (P＞0.05).The Tangshenping containing serum is abletoinhibit the proliferation of high glucose-induced epithelial cells of kidney tubules,and can reverse renal tubular-epithelial cell transdifferentiation via regulating RhoA/ROCK signaling pathway,and restrain renal interstitial fibrosis,thereby delaying the pathogenesis of diabetic kidney disease.
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Objective To investigate whether the apoptosis-2 ligand (Apo-2L),known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),could enhance irradiation-induced apoptosis in lung adenocarcinima H1975 cells that are resistant to the epithelial growth factor receptor (EGFR)-TKI.Methods Adenocarcinima H1975 cells were randomly divided into four groups:the control group,Apo2L group,irradiation group,and both Apo-2L and irradiation group.H1975 cells were pretreated with Apo2L under different concentrations of 200 and 228 ng/ml at 24 h before irradiation with doses of 1,1.5,2,2.5,3,3.5 and 4 Gy.The apoptosis rates of all groups were analyzed by flow cytometry 24 h post-irradiation.The inhibition rates of cell proliferation were measured by the MTT assay.Results MTT assay showed that the Apo-2L treatment significantly inhibited cell proliferation(x2 =136.17,P ＜ 0.05).The apoptosis rates of the four groups were different significantly,and the apoptosis rate of radiation combined with drug group was significantly higher than the other three groups(x2 =78.02,P ＜0.05).Conclusions The Apo-2L could not only inhibit the proliferation but also promote radiation-induced apoptosis of adenocarcinoma H1975 cells.
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BACKGROUND:Human glial cellline-derived neurotrophic factor (GDNF) and vascular endothelial growth factor 165 (VEGF 165 ) are essential genes for celldifferentiation. OBJECTIVE:To construct and identify pIRES 2-GDNF-VEGF 165 bicistronic eukaryotic expression vector. METHODS:Human GDNF genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. Then the GDNF cDNA fragment was inserted into the multiple cloning sites of pIRES 2-EGFP, to generate the bicistronic eukaryotic expression plasmid pIRES 2-GDNF-EGFP. The VEGF 165 gene was obtained from pIRES 2-VEGF 165-EGFP plasmid by twin PCR. Then VEGF 165 cDNA fragment was cloned into the pIRES 2-GDNF-EGFP, instead of EGFP, to create a double gene co-expressing vector plasmid pIRES 2-GDNF-VEGF 165 containing internal ribosome entry sites. Then pIRES 2-GDNF-VEGF 165 was used to transfect HEK293 cells. RT-PCR and western blot analysis were performed to test the co-expression of double genes. RESULTS AND CONCLUSION:DNA sequencing analysis demonstrated that the GDNF and VEGF 165 were exactly consistent with the sequence recorded in the GenBank. The size of GDNF gene was 636 bp and the size of VEGF165 gene was 576 bp. Enzyme digestion analysis indicated that, pIRES2-GDNF-VEGF165 bicistronic eukaryotic expression vector inserted GDNF band by Bgl II/Bam HI, inserted IRES-VEGF 165 fragment by Bam HI/Not I, and inserted GDNF-IRES-VEGF165 fragment by Bgl II/Not I. RT-PCR and western blot analysis showed that, after HEK293 cells were transfected with pIRES 2-GDNF-VEGF 165 , double genes were expressed at the mRNA and protein levels. The pIRES 2-GDNF-VEGF 165 bicistronic eukaryotic expression vector is successful y constructed.
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BACKGROUND:A series of studies indicate that autophagy is closely linked with differentiation. Bone morphogenetic protein 2 (BMP-2) is the classical pathway for C2C12 and MC3T3-E1 osteoblast differentiation, and the ideal model to study osteogenic differentiation process. OBJECTIVE:To observe the relationship between autophagy and BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation. METHODS:Real-Time PCR was applied to detect osteogenic differentiation and autophagy related index after C2C12 and MC3T3-E1 were induced with BMP-2 (100μg/L) for 72 hours. The osteogenic index alkaline phosphatase in BMP-2-induced MC3T3-E1 and C2C12 cultured with different concentrations of 3-methyladenine (0, 1, 5, 10 mmol/L) was determined with alkaline phosphatase staining. Western blot analysis was applied to detect LC3-I/II expression levels in C2C12 and MC3T3-E1 induced with BMP-2 for different time points (0, 12, 24, 48, 72, 96 hours). RESULTS AND CONCLUSION:The autophagy-related mRNA and protein expression showed an increasing tendency and autophagy-related protein LC3 levels was increased, which was associated with the time, during the BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation. Meanwhile, alkaline phosphatase expression levels were inhibited by autophagy in the process of osteogenic differentiation. Therefore, there is a close relationship between autophagy and the BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation.
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BACKGROUND:Anticancer drug and organic metal complexes wil form a new structure or a change in ion concentration, thus changing both the activity and toxicity to produce a synergistic effect. OBJECTIVE:To synthesize new high-efficient and low-toxic metal-fluorouracil complexes as anticancer drugs. METHODS:Copper, zinc and iron salts and fluorouracil were used to synthesize four copper, zinc and iron-fluorouracil complexes that were [Cu(5-Fu)2Cl2], [Cu(5-Fu)2(NO3)2], [Fe(5-Fu)3]SO4 and [Zn(5-Fu)2Cl2]. Preliminary chemical structures of the four complexes were confirmed by elemental analysis and mass spectrometry. Their inhibitory activity on human cancer cells, human leukemia cellline K562 and human colon cancer cellline HCT-116, was measured by MTT colorimetric assay. RESULTS AND CONCLUSION:[Cu(5-Fu)2Cl2], [Cu(5-Fu)2(NO3)2], [Zn(5-Fu)2Cl2] and [Fe(5-Fu)3SO4] were successful y synthesized. These four complexes at a mass concentration of 0.1-100 mg/L inhibited the proliferation of K562 and HCT-116 to different extents. The IC 50 values of these four complexes on K562 and HCT-116 cells were lower than those of fluorouracil, and their cytotoxicity was 1.5-7.8 times higher than that of fluorouracil. To conclude, copper/iron/zinc-fluorouracil complexes exhibit synergic inhibitory effects on cancer cellproliferation.
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Objective Observe the influence on the Gilial cell-line derived neurotrophic factor (GDNF) after treatment stroke rats with Bone marrow mesenchymal stem cells (BMSCs) or Bone marrow mononuclear cells (BMNCs)combined with traditional Chinese medicine (TCM).Methods Separating and cultivating BMSCs and BMNCs were.140 Wistar rats were divided into 7 groups randomly,normal group,pretended surgery group,model group,BMNC group,BMNC+TCM group,BMSC group,BMSC+TCM group.The other five groups were performed for 2 hours middle cerebral arterial occlusion (MCAO) except normal group and pretended surgery group.Intervention methods in each group after 24 hours of MCAO:model group:Subarachnoid injection of 100 μl 0.01M PBS,BMNC group and BMNC + TCM group,Subarachnoid injection of 2 × 107 BMNCS,BMSC group and BMSC+TCM group,Subarachnoid injection of 2 × 107 BMSCS,BMNC +TCM group and BMSC+TCM group were treated united with TCM on the transplantation day (po.Qd.).GDNF level in all groups' rat brain were analyzed by Enzyme-linked immuno sorbent assay (ELISA) method at the 4th day and the 28th day after transplantation.Results The GDNF level of model group [(62.60±4.05) pg/ml] is higher than normal group's [(53.46 ± 3.91)pg/ml] at the 28th day (P＜ 0.05).The GDNF levels of BMNC group [(194.21 ±39.56)pg/ml,(67.70±4.73)pg/ml] and BMSC group [(169.83±28.84)pg/ml,(82.66±32.23)pg/ml] are higher than model group's at the 4th and 28th day(P＜0.05).The GDNF level of BMSC group is higher than BMNC group's at the 28th day(P＜0.05).The GDNF levels of group BMNC+TCM group[(560.61 ± 194.84) pg/ml,(265.83 ±93.58) pg/ml and BMSC+TCM group[(370.93 ±46.19) pg/ml,(247.34±98.02)pg/ml] are higher significantly than BMNC group's or BMSC group's at the 4th and 28th day(P＜0.05).At the 4th day the GDNF level of the BMNC+TCM group is higher than BMSC+TCM group's(P＜0.05).Conclusion Subarachnoid transplantation of BMNCs or BMSCs will increase the GDNF level in brain of MCAO rats.The transplantantion combined with TCM can inprove the capability of the enhance.That reflect the advantage of transplantantion bone marrow origin stem cell united with TCM.
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Objective: To establish a metastatic cell line from distant organ metastasis using Mc3 cell line in nude mice. Methods: Tail vein injection of Mc3 cells and cell culture technic were employed to induce metastasis in distant organ . Cell counting and flow cytometry were used to study the cell growth. Karyotype analysis and histopathological observation were used to study the morphological features with light and electron microscopy. Results: Paralized nude mouse was observed in 1 out of 50 experimental nude mice. The cells derived from the spinal cord were cultured and transferred for more than 50 passages. The cells were proved to be of mucoepidermoid carcinoma from human being by the morphology, histopathology and karyotype of the cells. The population doubling time and S-phase cell of the cells were 43 h and 22.7% respectively. The cell line was named Ms. Conclusion: Ms is a metastatic cell line of spinal cord metastasis in nude mouse derived from human mucoepidermoid cacinoma cells.
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Objective To explore the differentiation potential of bone marrow stromal cells (BMSC) to enteric neuron in vitro and to seek proper induction methods.Methods BMSC were harves- ted from male rats and cultured in DMEM supplemented with 20% fetal bovine serum,and characterized by flow cytometry.At passage 6,BMSC were pre-induced by basic fibroblast growth factor (bFGF,10 ng/ml) for 24 h,then induced in two groups:glial cell-line derived neurotrophic factor (GDNF) group, 10 ng/ml GDNF in fetal gut condition medium (FGCM) for 10 d.Vitamin A acid (VA) group,VA, zinc in FGCM for 10 d.The expressions of neuronal markers,neural specific enolase (NSE) and neu- rofilament (NF),glial cell marker,glial fibrillary acidic protein (GFAP),enteric neuronal marker,pro- tein gene production 9.5(PGP9.5),nitric oxide synthase (nNOS),enteric neural transmitter,vasoactive intestinal peptide (VIP) were detected by fluorescent immunohistochemistry method.Results The cul- tured BMSC were CD90 positive and CD45 negative on flow cytometry.After 10 d of induction,a certain number of cells adopted neuron-like morphological changes and showed the expressions of NSE,NF, PGPg.5,nNOS and VIP without the expression of GFAP by fluorescent immunohistoehemistry method in both groups.But in GDNF group,the positive rate of NF,PGP9.5,nNOS and VIP was significantly higher than that in VA group (75.6%?8.4% vs 48.5?7.5%;57.7%?6.5% vs 35.7%?7.2% 46.6%?5.4% vs 30.5%?6.6%;72.3%?6.7% vs 40.4%?7.4%;P＜0.01).Conclusion BMSC can be induced to differentate into enteric neuron in vitro by different methods.GDNF with FGCM can induce higher rate of enteric neuron like cells compared with VA etc.
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Objective To examine the effectiveness of a gene transfer of rat glial cell-line derived neurotrophic factor(GDNF)into Schwann cells Methods Rat GDNFcDNA was amplified from newly-born rat sciatic nerve by RT-PCR and ligated into the retroviral vector pLNCX-2 A fter being packaged by the amphotropic packaging cell line PT67,the viral supernatants from these anti-clonal producing lines were titred on fibroblast N IH3T3 The highest tire recombinant retrovirus was used to infect deviding populations of newly-born rat SCs The level of GDNF mRNA in GDNF-SCs and normal SCs was tested by RT-PCR The GDNF protein in genetically modified SCs and normal SCs was assayed by immunocytochemistry The amounts of GDNF in conditioned medium of GDNF-SCs in different phrase and normal SCs were detected by enzyme-linked immunoassay sensitive assay Biological activity of the secreted GDNF was tested using motoneuron bioassay by MTT method Results (1)The GDNF cDNA band of GDNF-SCs was more prominent than that of SCs (2)Cells in both groups were immunohistochemically positive for GDNF expression Staining for GDNF was more prominent in the Scs infected with pLNCX2-GDNF (3)The rate of GDNF secreted by GDNF-Scs was 5 1-fold compared with normal SCs (4)The bioassay comfirmed that the secreted GDNF from GDNF-Scs was biologically active,and more motoneurons survived than normal SCs group Conclusion With the help of retrovirus,GDNF gene can be transferred and stably expressed in SCs,and it′s may be a better way to graft SCs promoing regeneration of PNS injuries.