人脐带间充质干细胞分泌物对肝细胞增殖和凋亡的影响

Objective To investigate the effect of human umbilical cord mesenchymal stem cell paracrine substance on proliferation and apoptosis of liver cells in vitro. Methods Mesenchymal stem cells (MSC)were separated from human umbilical cord with type Ⅳ collagenase and trypsogen digestion method and cultured in vitro. The human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM) which contain paracrine substance of human umbilical cord mesenchymal stem cells (HUCMSC) was prepared. Hepatocytes were isolated from SD rats by low concentration collagenase perfusion procedure. There were three groups in the experiment, control group, 2% MSC-CM group and 8% MSC-CM group. The proliferation of normal hepatocytes were assayed with MTT method. We detected the urea and albumin level in culture supernatant to assay the hepatocyte function under different concentration MSC-CM. Hepatocytes were induced for apoptosis by Actinomycin D and tumor necrosis factor alpha (TNF-α),and the apoptosis effect of different concentration MSC-CM was assayed with LIVE/DEAD Viability/Cytotoxicity Kit. Results The MTT assay showed that the absorbance of 2% MSC-CM group was significantly increased (P＜0. 01), and the urea and albumin levels of 2 % MSC-CM group were also significantly increased when compared with control group(P＜0. 01).LIVE/DEAD Viability/Cytotoxicity Kit revealed that hepatocyte survival rate of 2 % MSC-CM group was increased when compared with control group(P＜0. 05), there were no significant differences in above-mentioned experiments when 8% MSC-CM group compared with control group. Conclusion The low concentration MSC-CM could stimulate normal hepatocyte proliferation, inhibit impaired hepatocyte apoptosis and improve hepatocyte function.     

人脐带间充质干细胞分泌物对肝细胞增殖和凋亡的影响

Objective To investigate the effect of human umbilical cord mesenchymal stem cell paracrine substance on proliferation and apoptosis of liver cells in vitro. Methods Mesenchymal stem cells (MSC)were separated from human umbilical cord with type Ⅳ collagenase and trypsogen digestion method and cultured in vitro. The human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM) which contain paracrine substance of human umbilical cord mesenchymal stem cells (HUCMSC) was prepared. Hepatocytes were isolated from SD rats by low concentration collagenase perfusion procedure. There were three groups in the experiment, control group, 2% MSC-CM group and 8% MSC-CM group. The proliferation of normal hepatocytes were assayed with MTT method. We detected the urea and albumin level in culture supernatant to assay the hepatocyte function under different concentration MSC-CM. Hepatocytes were induced for apoptosis by Actinomycin D and tumor necrosis factor alpha (TNF-α),and the apoptosis effect of different concentration MSC-CM was assayed with LIVE/DEAD Viability/Cytotoxicity Kit. Results The MTT assay showed that the absorbance of 2% MSC-CM group was significantly increased (P＜0. 01), and the urea and albumin levels of 2 % MSC-CM group were also significantly increased when compared with control group(P＜0. 01).LIVE/DEAD Viability/Cytotoxicity Kit revealed that hepatocyte survival rate of 2 % MSC-CM group was increased when compared with control group(P＜0. 05), there were no significant differences in above-mentioned experiments when 8% MSC-CM group compared with control group. Conclusion The low concentration MSC-CM could stimulate normal hepatocyte proliferation, inhibit impaired hepatocyte apoptosis and improve hepatocyte function.     

Effect of Ureaplasma urealyticum infection on IL-laand IL-6 secretion by rat Sertoli cells

Objective: To observe the effect of Ureaplasma urealyticum (UU) infection on the IL-1α and IL-6 secretion by rat Sertoli cells. Methods: Eight 20-day-old UU-free male SD rats (average weight 40 g) were used. Under sterile condition, the testes were removed and separately digested with collagenase typeⅡand hyaluronidase. High purity Sertoli cells were then isolated and adjusted to a concentration of 8×105/mL with DMEM/Ham's F-12. In the infected group, 1 mL Sertoli cell suspension and 100 mL UU (serotype 8, T960) were introduced into one well of a 24 well culture plate. In the control group, 1 mL Sertoli cell suspension and 100 mL medium were introduced. IL-1αand IL-6 were determined in the culture supernatant with ELISA. Results: The production of IL-1αwas significantly lower and of IL-6 significantly higher in the infected than those in the control groups (P<0.01). Conclusion: UU infection reduces the IL-1αand increases the IL-6 secretion by rat Sertoli cells. UU infection is probably involved in     

急性胰腺炎胰腺腺泡细胞胀亡和巨噬细胞激活的关系

Objective To study the relationship between the oncosis of pancreatic acinar cells and activa-tion of macrophage in rat model of acute pancreatitis (AP). Methods The pancreatic acinar cells were isolated by two-step enzyme digestion, and then they were divided into control group, AP group and test group. Pancreatic acinar cells were cultured with caerulein in AP group, with caerulein and endothelin in test group, and with culture medium in control group. The oncesis rate of the pancreatic acinar cells was detected after acridine orange and ethidium bromide fluorescent staining. The supernatant was collected to detect the release of amylase and lactate dehydrogenase (LDH). The macrophages were cultured with 1 ml of supematant for 6 hours, and then the protein level of tumor necrosis factor-α (TNF-α) was measured by ELISA. Results Few oncotic pancreatic acinar cells were observed in the control group, and the levels of amylase and LDH secreted by pancreatic acinar cells and TNF-α secreted by macrophage were (1175±165)kU/L, (846±118)U/L and (36±5)μg/L, respectively. Oncotic pancreatic acinar cells were observed in AP group, and the levels of amylase, LDH and TNF-α were (7130±680) kU/L, (4262±626) U/L and (155±18) μg/L, respectively, which were significantly higher than those in control group (t = 5.184, 4.277, 3.665, P < 0.05). The levels of amylase, LDH and TNF-α were even higher in test group, and they were (9240±1177) kU/L, (6937±893)U/L and (268±35)μg/L, respectively, which were significantly higher than those in AP group (t = 2.251, 2.825, 2.843, P < 0.05). Conclusions The release of amylase was changed as the oncosis of pancreatic acinar cells occurred. The secretion of TNF-α was along with the degree of oncosis of pancreatic acinar cells. The results of the study indicate that a relationship exists among the inflammatory response of macrophage, the release of contents of pancreatic acinar cells and the oncosis of the pancreatic acinar cells.     

Bone regeneration with osteogenic matrix cell sheet and tricalcium phosphate:An experimental study in sheep

AIM To determine the effects of a cell sheet created from sheep bone marrow and tricalcium phosphate(TCP) on osteogenesis.METHODS Bone marrow cells were harvested from a sheep and cultured in a minimal essential medium(MEM) containing ascorbic acid phosphate(AscP) and dexamethasone(Dex). After 2 wk, the formed osteogenic matrix cell sheet was lifted from the culture dish using a scraper. Additionally, harvested bone marrow cells were cultured in MEM only as a negative control group, and in MEM with AscP, Dex, and β-glycerophosphate as a positive control group. For in vitro evaluation, we measured the alkaline phosphatase(ALP) activity and osteocalcin(OC) content in the media of the cultured cells from each group. For in vivo analysis, a porous TCP ceramic was used as a scaffold. We prepared an experimental group comprising TCP scaffolds wrapped with the osteogenic matrix cell sheets and a control group consisting of the TCP scaffold only. The constructs wereimplanted subcutaneously into athymic rats and the cell donor sheep, and bone formation was confirmed by histology after 4 wk.RESULTS In the in vitro part, the mean ALP activity was 0.39 ± 0.03 mg/well in the negative control group, 0.67 ± 0.04 mg/well in the sheet group, and 0.65 ± 0.07 mg/well in the positive control group. The mean OC levels were 1.46 ± 0.33 ng/well in the negative control group, 3.92 ± 0.16 ng/well in the sheet group, and 4.4 ± 0.47 ng/well in the positive control group, respectively. The ALP activity and OC levels were significantly higher in the cell sheet and positive control groups than in the negative control group(P 0.05). There was no significant difference in ALP activity or OC levels between the cell sheet group and the positive control group(P 0.05). TCP constructs wrapped with cell sheets prior to implantation showed bone formation, in contrast to TCP scaffolds alone, which exhibited poor bone formation when implanted, in the subcutaneous layer both in athymic rats and in the sheep. CONCLUSION This technique for preparing highly osteoinductive TCP may promote regeneration in large bone defects.     

胰岛素对脂肪干细胞生长因子分泌功能的影响

Objective To study the effect of insulin in different concentrations on secretion function of growth factors of adipose-derived stem cells (ADSCs). Methods ADSCs were isolated from human ab-dominal adipose tissue and cultured. The immunophenotype and adipose induced-differenciation were identi-fied, and the third generation cells were collected. The collected cells were assigned to 1 × 10-8, 1 × 10-7, 1 × 10-6 mol/L insulin groups according to the concentration of added insulin. When cells grew into 70% confluence in conventional medium, ADSCs were cultured further in serum-free DMEM containing insulin in different concentrations for 3 days. ADSCs cultured in medium without insulin were used as control group. Secretion amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) of ADSCs were determined by enzyme-linked immunosorbent assay. The effects of the supernatant fluid of ADSCs' nutrient solution on the proliferation and collagen synthesis of the cultured fibroblast were detected by MTT chromatometry and hydroxyproline chromatometry. Results The secretion amounts of VEGF and HGF of ADSCs in 1 × 10-8 and 1 × 10-7 mol/L insulin groups [ (471±41, 762±66 ng/L), (643±64, 930±67 ng/L) , respectively ] were significantly higher as compared with those in control group (286±47, 577±84 ng/L) ( P <0.05 orP <0.01). No change occurred in the secretion amount of VEGF and HGF of ADSCs in 1±10-6 mol/L insulin group ( P >0.05 ). The supernatant fluid of ADSCs' nutrient medium of 1 ± 10-8 ,1 ± 10-7 mol/L insulin groups showed obvious stimulative effect on the proliferation and collagen synthesis of fibroblasts, and it was most obvious in the 1 ± 10-7 mol/L group ( P < 0.05 or P < 0. 01 ). Conclusions Insulin in the concentrations of 1 ± 10-8 and 1 ± 10-7 mol/L can notably promote ADSCs' function of secreting VEGF and HGF.     

RhoA基因沉默对肝癌HepG2细胞增殖和迁移能力的影响

Objective To construct a RhoA-siRNA expression vector and determine its role on the malig-nant behavior of HepG2 cells.Methods A RhoA-siRNA DNA fragment was synthesized and cloned into the expression vector of pGenesil-1.The constructed Rhon-siRNA DNA plasmid was stably transfected into HerG2 cells by lipofectamine,and then HepG2 cells were divided into the HepG2/RhoA-siRNA group (HepG2 cells were transfected with pGenesil-1-RhoA-siRNA),HepG2/control group(HepG2 cells were transfected with control plasmid) and HepG2 group (without plasmid transfection).The inbibitory effect of RhoA-siRNA on RhoA protein expression was shown by Western blot.The proliferation,migration,growth potentiality and cell cycle of transfected HepG2 cells were evaluated by MTT assay,wounded healing,the plate cloning formation test and flow cytometry,respectively.All data were analyzed by one-way analysis of variance (ANOVA) and chi-square test.Results The expression of RhoA protein in the HepG2/RhoA-siRNA group was,significantly decreased compared with that in the other two groups (F=178.19,P<0.05).Scratched cells were healed within 48 hours in the HepG2/control group and HepG2 group,but not in the HepG2/RhoA-siRNA group.The clone formation rates in the HepG2/RhoA-siRNA group,HepG2 group and HepG2/control group were 39%±3%,67%±5%and 70%±6%,respectively,with a significant difference among the three groups(χ2=33.34,38.69,P<0.05).Flow cytometry showed that the number of cells transfected with RhoA-siRNA was highest in the G0/G1 phase and lowest in the S phase(F=70.46,76.57.P<0.05).Conclusion The RhoA-siRNA expression vector can effectively suppress the proliferation and migration of HepG2 cells,which may provide a novel gene therapy for hepatocellular carcinoma.     

RhoA基因沉默对肝癌HepG2细胞增殖和迁移能力的影响

Objective To construct a RhoA-siRNA expression vector and determine its role on the malig-nant behavior of HepG2 cells.Methods A RhoA-siRNA DNA fragment was synthesized and cloned into the expression vector of pGenesil-1.The constructed Rhon-siRNA DNA plasmid was stably transfected into HerG2 cells by lipofectamine,and then HepG2 cells were divided into the HepG2/RhoA-siRNA group (HepG2 cells were transfected with pGenesil-1-RhoA-siRNA),HepG2/control group(HepG2 cells were transfected with control plasmid) and HepG2 group (without plasmid transfection).The inbibitory effect of RhoA-siRNA on RhoA protein expression was shown by Western blot.The proliferation,migration,growth potentiality and cell cycle of transfected HepG2 cells were evaluated by MTT assay,wounded healing,the plate cloning formation test and flow cytometry,respectively.All data were analyzed by one-way analysis of variance (ANOVA) and chi-square test.Results The expression of RhoA protein in the HepG2/RhoA-siRNA group was,significantly decreased compared with that in the other two groups (F=178.19,P<0.05).Scratched cells were healed within 48 hours in the HepG2/control group and HepG2 group,but not in the HepG2/RhoA-siRNA group.The clone formation rates in the HepG2/RhoA-siRNA group,HepG2 group and HepG2/control group were 39%±3%,67%±5%and 70%±6%,respectively,with a significant difference among the three groups(χ2=33.34,38.69,P<0.05).Flow cytometry showed that the number of cells transfected with RhoA-siRNA was highest in the G0/G1 phase and lowest in the S phase(F=70.46,76.57.P<0.05).Conclusion The RhoA-siRNA expression vector can effectively suppress the proliferation and migration of HepG2 cells,which may provide a novel gene therapy for hepatocellular carcinoma.     

血小板源性生长因子BB对大鼠肌腱愈合和肌腱粘连的影响

目的 了解血小板源性生长因子BB(PDGF-BB)基因转染大鼠肌腱细胞对肌腱愈合及肌腱粘连的影响. 方法 将90只SD大鼠制成跟腱损伤模型,按随机数字表法分为3组,每组30只:实验组,肌腱断端注射20μL转染PDGF-BB基因的大鼠肌腱细胞(1×108个/mL);对照组,肌腱断端注射20μL未行转染的大鼠肌腱细胞(1×108个/mL);空白对照组,不做任何处理.6-0丝线行改良Kessler法缝合跟腱,管型石膏固定1周.通过基因测序及RT-PCR鉴定转染PDGF-BB基因的大鼠肌腱细胞.分别于术后3 d和1、2、4、8周取各组大鼠肌腱组织样本,行大体、组织学观察以及生物力学检测,对比各组肌腱粘连度、组织中Fb数量与胶原纤维含量、肌腱最大抗拉力及最大滑动距离、组织中PDGF-BB的浓度.对数据行t检验. 结果(1)转染的肌腱细胞经RT-PCR以及基因测序证实在体外稳定表达PDGF-BB mRNA.(2)各组大鼠术后3 d肌腱均出现较明显肿胀及炎性细胞浸润,实验组改变较其他组明显轻微;随后各组情况均逐渐好转.术后4、8周肌腱粘连度分级组间比较未见明显差异.(3)实验组Fb数在术后2、4、8周显著低于对照组和空白对照组(t值分别为2.94、4.26、5.76和4.00、3.83、6.12,P＜0.05或P＜0.01).(4)实验组术后4周胶原纤维含量为(43±6)%,较对照组[(55±8)%]与空白对照组[(61±8)%]显著下降(t值分别为2.94和4.41,P＜0.05或P＜0.01).(5)术后4、8周实验组肌腱最大滑动距离为(3.25±0.33)、(3.65±0.21)mm,显著高于对照组的(2.29±0.40)、(2.21±0.37)mm和空白对照组的(2.01±0.23)、(1.89±0.24)mm(t值分别为4.53、8.29和7.55、13.52,P值均小于0.01),但其肌腱最大抗拉力与另2组比较差异无统计学意义(t值分别为0.41、0.41和0.77、0.72,P值均大于0.05).(6)术后3 d和2、4周,实验组肌腱组织中PDGF-BB浓度为(12.95±1.36)、(8.32±0.94)、(9.10±1.06)ng/mL,均显著高于对照组的(1.13±0.21)、(2.07±0.48)、(3.85±0.39)ng/mL(t值分别为21.04、14.50、11.39,P值均小于0.01). 结论 转染PDGF-BB基因肌腱细胞有促进肌腱内源性愈合、减轻肌腱粘连的作用.     

水蛭素抑制增生性瘢痕成纤维细胞的实验研究

Objective To study the effect of hirudin on the function of human hyperplastic scar fi-broblasts (HSFBs). Methods HSFBs were cultured in vitro. Hirudin solution in the concentration of 1, 10, and 50 kU/L was respectively added into DMEM culture medium to form 1, 10, and 50 kU/L hirudin groups, with 9 wells in each group. HSFBs cultured without hirudin were set up as control group. Cell inhi-bition rate, secretion level of TGF-β1 from cells, and expression levels of mRNA of type Ⅰ and Ⅲ precolla-gem were determined at 24, 48, and 72 h after culture. Results Inhibition rates of HSFBs growth was re-spectively ( 29.3 ± 0.9) % , ( 30.1 ± 0.3 ) % , and (45.2 ± 1.9 ) % when cultured with 10 kU/L hirudin for 24, 48, and 72 hs, which were higher than those in control group [ (0.0±0.0)% , P <0.05]. There was statistically significant difference between control group and 1 and 50 kU/L hirudin groups in the inhibition rates of HSFBs at some time points ( P <0.05). Secretion level of TGF-β1 of HSFBs in 1, 10, 50 kU/L hirudin groups was respectively (228.5±1.8), (210.5±11. 1), and (168.5±14.1) pg/mL when cul-tured for 48 hs, of which the last 2 figures were significantly lower than that of control group [ (265.0±1.5) pg/mL, P < 0.05 ]. Hirudin in the concentration of 10 and 50 kU/L could inhibit the expression of mRNA of type Ⅰ and Ⅲ precollagen in HSFBs. Conclusions Hirudin solution in the concentration of 10 and 50 kU/L can inhibit the proliferation of HSFBs and secretion of TGF-β1 and collagen in certain degree.     

肝细胞生长因子对人外泌汗腺上皮细胞促增殖作用的研究

目的 研究肝细胞生长因子(hepatocyte growth factor, HGF)对培养的人外泌汗腺上皮细胞(human eccrine sweat gland epithelial cells, hESGc)的促增殖作用,以及细胞内磷酸化细胞外信号调节激酶(phosphorylated extracellular signal-regulated kinase 1/2, P-ERK1/2)蛋白的表达.方法 在无血清角质形成细胞培养基(keratinocyte serum free medium, KSFM)中培养hESGc,取第1代细胞进行实验.免疫组织化学染色检测C-met表达.MTT法检测HGF对hESGc的促增殖作用,实验分为3组:空白组、对照组和实验组.空白组仅含200μLKSFM;对照组于96孔板以2×103个/孔接种hESGc,并加入200μLKSFM;实验组于96孔板以2×103个/孔接种hESGc并加入200μLKSFM后,再分别加入不同浓度(2、20、40、80ng/mL)HGF.实验组于加入各浓度HGF前以及加入后2、4d,观察细胞增殖情况.实验组于加入40ng/mL HGF后即刻,5、30、90和120min,采用Western blot方法检测细胞内p-ERK1/2表达规律.结果 hESGc抗-C-met免疫组织化学染色胞浆可见阳性染色,细胞核染色为蓝色.MTT法检测示40、80ng/mLHGF对hESGc具有显著促增殖作用(P<0.05).在含40ng/mL HGF的KSFM中培养2d,吸光度(A)值为0.2393±0.0709,增殖率为74.2%,对照组A值为0.1374±0.0290;培养4d,40ng/mL HGF实验组A值为0.2878±0.0743,增殖率为74.8%,对照组A值为0.1646±0.0350,两组比较差异均有统计学意义(P<0.05).在含80ng/mL HGF的KSFM中培养2 d,A值为0.2123±0.0592,增殖率为54.5%;培养4d,80ng/mL HGF实验组A值为0.2310±0.0567,增殖率为40.3%;与对照组比较差异均有统计学意义(P<0.05).p-ERK1/2在40ng/mL HGF刺激5min后表达达高峰,相对积分吸光度值(relative integral absorbance, RIA)为0.592.±1922,较刺激后即刻增加8.1倍(P<0.01);刺激30、90及120min后RIA与刺激后即刻比较,差异无统计学意义(P>0.05)结论 HGF可促进hESGc增殖,并能引起ERK1/2蛋白磷酸化.     

体外诱导毛囊干细胞成血管平滑肌细胞的实验研究

目的探讨体外诱导人毛囊干细胞成血管平滑肌细胞的可行性。方法采用中性蛋白酶(Dispase)分离人毛囊干细胞,用含10 ng/mL PDGF-BB、10%血清的低糖DMEM诱导液对其进行诱导,无PDGF-BB的培养液为对照组,观察每代细胞形态,诱导4代后检测α-平滑肌肌动蛋白(α-SM actin)与肌钙结合蛋白(Calponin)的表达。结果在诱导液的作用下,细胞形态逐步向平滑肌样转变,对照组细胞形态改变不显著。细胞免疫荧光检测显示,至第4代,实验组已明显表达α-SM actin与Calponin,对照组表达不明显;流式细胞仪检测显示,实验组α-SM actin与Calponin阳性表达率近50%,对照组则低于8%;RT-PCR显示,实验组表达Calponin,而对照组未见明显表达。结论使用含10 ng/mLPDGF-BB的低糖DMEM培养液可体外诱导人毛囊干细胞成血管平滑肌细胞。     

转染血红素氧合酶—1基因减轻人肝细胞体外缺氧—复氧损伤

目的 探讨血红素氧合酶-1重组腺病毒载体(Ad-HO-1)体外转染人肝细胞的转染效果及其对肝细胞缺氧-复氧损伤的影响.方法 取人肝细胞系L-02细胞,滴加低温保存的Ad-HO-1,分别培养24 h、48 h和72 h(24 h组、48 h组和72 h组),并以加入空载体腺病毒共培养的L-02细胞为空白对照.采用逆转录聚合酶链反应法检测各组肝细胞HO-1 mRNA的表达水平;以间接免疫荧光标记法检测各组肝细胞的HO-1表达率;在倒置荧光显微镜下观察转染24 h和72 h的肝细胞中绿色荧光蛋白(EGFP)的表达.取空白对照组肝细胞(培养48 h)和48 h组肝细胞,缺氧培养4 h后再有氧培养8 h,采用四甲基偶氮唑盐法测定两组肝细胞存活率.结果 24 h组、48 h组和72 h组HO-1 mRNA表达水平明显高于空白对照组,且随着转染时间的延长,HO-1 mRNA的表达水平逐渐升高.空白对照组HO-1的表达率为2.0%,24 h组为29%,48 h组为85.6%,72 h组为84.6%.基因转染后24 h和72 h,可以观察到L-02细胞中EGFP的表达.经历缺氧-复氧实验后,空白对照组肝细胞的存活率为(37.7±3.5)%,48 h组肝细胞的存活率为(89.4±5.2)%,二者相比较,差异有统计学意义(P<0.01).结论 Ad-HO-1在体外能有效的转染人肝细胞;与未转染者相比,转染肝细胞的缺氧-复氧损伤程度较轻.     

脂肪干细胞促进人表皮角质形成细胞创面模型愈合的研究

目的 探讨脂肪干细胞(ADSCs)促进人表皮角质形成细胞(HEKa)创面模型愈合的可能性.方法 体外培养大鼠ADSCs(rADSCs)(n=10).实验组为rADSCs与HEKa直接共培养组(n=10),对照组为在transwell培养板建立的rADSCs与HEKa间接共培养(间接组,n=8)和单纯HEKa培养组(单纯组,n=8).刮擦生长融合成片的HEKa细胞制备创面.培养24、48、72 h后,计数迁移到创面的HEKa细胞数量并计算创面愈合率,检测HEKa细胞吸收脱氧胸腺嘧啶核苷的放射活性以判定HEKa细胞的增殖活性.结果 实验组、间接组、单纯组在伤后24 h分别有(9.2±0.2)、(5.0±0.3)、(4.2±0.3)个细胞/高倍视野;48 h分别有(58.5±0.4)、(26.5±0.3)、(20.7±0.5)个细胞/高倍视野;72 h分别有(125.8±0.4)、(43.0±0.5)、(35.6±0.5)个细胞/高倍视野越过创缘进入创面,实验组均明显优于两对照组(P<0.05).三组创面愈合率在伤后72 h分别为61.0% ±3.0% 、35.0% ±2.5% 、32.0% ±2.1% ,实验组均明显优于两对照组(P<0.05).脱氧胸腺嘧啶核苷掺入培养基法表明三组HEKa细胞的放射性强度为(1440±210)、(1050±280)、(1130±390)cpm/105细胞,实验组与两对照组之间差异有统计学意义(P<0.05).结论 rADSCs通过直接接触促进HEKa细胞的分裂增殖和迁移.     

碱性成纤维细胞生长因子在富集结直肠癌肿瘤干细胞中的作用

目的 研究不同浓度碱性成纤维细胞生长因子（bFGF）作用于结直肠癌细胞不同时间,富集肿 瘤干细胞的效果。方法? 结直肠癌DLD-1细胞分别在含5 ng/mL、10 ng/mL、20 ng/mL bFGF的无血清培 养基中悬浮培养,分为G1、G2、G3组,设置培养时间梯度为10 d、20 d、30 d,获得球细胞。采用流式 细胞术（FCM）检测细胞球中的CD44+、CD133+及CD44+CD133+双阳性的细胞表达比例,Real-time PCR 检测球细胞中的干细胞基因（KLF4、Nanog）;上皮间质转化基因（E-cadherin、Snail）;Wnt/β-catenine通路 基因（Wnt-3a）的 mRNA表达情况,成球实验检测细胞球的自我更新能力。结果 FCM检测结果：CD44+ 阳性表达的细胞表达以G2组20 d最高,CD133+及CD44+CD133+双阳性的细胞表达均以G2组20 d及G3 组10 d最高,差异有统计学意义（F=98.895、147.641、13.321,P<0.05）。 Real-time PCR检测结果：各组中 KLF4、Nanog、Snail以及Wnt-3a mRNA的表达均以G2组20 d表达最高（F=2.424、7.694、2.951、3.771, 均P<0.05）, E-cadherin基因G2组20 d mRNA表达最低（G2组30 d、G1组10 d除外）（F=10.620,P<0.05）。 成球实验结果：各组比较G1组20 d和G2组20 d成球数量明显多于其他组,差异具有统计学意义（F=14.279, P<0.01）;但 G1组20 d和G2组20 d比较,无统计学差异（t=0.605,P=0.578）。 结论 碱性成纤维细胞生长 因子无血清悬浮培养能够有效富集肿瘤干细胞,其中G2组培养20 d较其余组能更有效地富集结直肠癌肿 瘤干细胞。     

类胰岛素生长因子—1作用下肌腱细胞的周期改变

为了明确类胰岛素生长因子－１（ＩＧＦ－１）促进肌腱细胞生长的作用机制，以便进一步探索调控肌腱细胞生长的手段，采用体外培养的第６代肌腱细胞，加入ＩＧＦ－１共同培养后，与对照组一起，用流式细胞技术进行细胞周期亚时相的定量分析。结果表明，实验组的ＤＮＡ合成前期（Ｇ１期）时间、ＤＮＡ合成期（Ｓ期）时间和分裂前期及分裂期（Ｇ２Ｍ期）时间分别为１１．８，２１．４和６．８小时；而对照组的时间分别为２５．６，２２．６和２１．８小时。ＩＧＦ－１使肌腱细胞的Ｇ１期和Ｇ２Ｍ期所需要的时间大大缩短。说明ＩＧＦ－１对肌腱细胞生长的促进作用是通过加快Ｇ１期和Ｇ２Ｍ期的进程实现的