人脐静脉内皮细胞胰岛素抵抗细胞模型的建立

背景：有研究表明血管内皮可能是胰岛素抵抗发生的首要环节。 目的：采用高浓度胰岛素体外诱导培养法建立人脐静脉内皮细胞胰岛素抵抗模型。 方法：以人脐静脉内皮细胞系为研究对象,应用含不同浓度胰岛素(50,40,30,20,10,1,0.1,0.01 U/L)的DMEM培养基与人脐静脉内皮细胞共同培养12,24,36,48,72 h,并设置正常组,光学显微镜下观察细胞形态学变化;MTT法测定各组细胞活性;用葡萄糖氧化酶-过氧化物酶法法鉴定胰岛素抵抗细胞模型。 结果与结论：与正常组比较,当胰岛素浓度大于40 U/L,其作用时间大于48 h时,细胞形态受损严重,细胞活性显著下降(P < 0.01);当胰岛素浓度小于20 U/L,作用时间小于36 h时细胞形态无明显损伤(P > 0.05)。葡萄糖氧化 酶-过氧化物酶法实验表明,在30 U/L的胰岛素刺激48 h条件下,培养液中残存葡萄糖浓度显著高于正常组细胞 (P < 0.01)。证实,用含30 U/L胰岛素的培养基培养48 h的人脐静脉内皮细胞细胞可产生胰岛素抵抗。     

虎杖苷对成纤维细胞生物学特性的影响

背景：虎杖多年来一直是烧伤、创伤创面愈合治疗方剂中的一味主药,因成分复杂,具体药理作用难以进一步研究,对于虎杖苷促愈合作用目前未见文献报道。 目的：分析不同浓度虎杖苷对成纤维细胞生物学特性的影响。 方法：取烧伤后行瘢痕切除植皮4例患者剩余小中厚皮片,原代培养人成纤维细胞。用含有10^-6,10^-5,10^-4,10^-3,10^-2 mol/L不同浓度虎杖苷培养液作用第2代人成纤维细胞,未加虎杖苷的培养液作为对照。MTT法检测细胞增殖情况;流式细胞仪检测细胞周期及凋亡;ELISA法检测上清中纤维结合蛋白、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白的表达情况。 结果与结论：10^-5、10^-4 mol/L组促进成纤维细胞增殖最明显,10^-2 mol/L组吸光度值显著下降,细胞生长受抑制。10^-3 mol/L组G1期细胞大幅度下降,细胞有S期阻滞现象。10^-2 mol/L组有明确的促凋亡作用。10^-2 mol/L组上清中Ⅰ、Ⅲ型胶原蛋白较其他各组显著增高(P < 0.05);纤维结合蛋白较对照组显著增高(P < 0.05),较其他各组有所下降(P < 0.05)。说明低浓度虎杖苷有促进成纤维细胞增殖、保护细胞免于凋亡及促进纤维结合蛋白的表达与合成分泌的作用,促进成纤维细胞增殖的最适浓度应为10^-5~10^-4 mol/L。     

转录因子2过表达改善人肝癌细胞HepG2的胰岛素抵抗

目的研究转录因子2(TCF2)过表达对HepG2细胞胰岛素抵抗的影响。方法用高浓度胰岛素(1×10-8mol/L)诱导处理人肝癌HepG2细胞24 h建立人肝癌HepG2细胞胰岛素抵抗模型,分为空白组、胰岛素抵抗模型组(IR组)、IR+空载体组和IR+TCF2过表达组;RT-qPCR和Western blot检测TCF2的表达;葡萄糖氧化酶法检测培养液中的葡萄糖浓度;蒽酮法检测糖原合成量;MTT法检测细胞存活率;比色法检测己糖激酶和丙酮酸激酶的活性;Western blot检测人胰岛素受体底物(IRS-1)和葡萄糖转运蛋白4(GLUT4)的表达。结果与空白组比较,IR组细胞葡萄糖消耗量显著降低(P<0.05),表明模型建立成功。IR组细胞TCF2 mRNA和蛋白表达显著降低(P<0.05)。与IR组相比,TCF2过表达可显著增加胰岛素抵抗HepG2细胞的葡萄糖消耗量、肝糖原合成量、己糖激酶活性和丙酮酸激酶活性,增加IRS-1和GLUT4蛋白表达(P<0.05)。结论过表达TCF2可改善人肝癌HepG2细胞的胰岛素抵抗。     

葡萄糖浓度与大鼠胰腺导管干细胞的诱导分化

背景：葡萄糖是胰腺导管干细胞分化的重要因素之一,与分化后胰岛素分泌细胞数量及分泌能力相关。 目的：对比不同浓度葡萄糖诱导下,胰腺导管干细胞分化后细胞的胰岛素分泌能力。 方法：使用胶原酶Ⅴ及Ficoll-400分离及纯化Wistar大鼠胰腺上皮细胞,获取胰腺导管干细胞,将干细胞分为10组,体外培养、增殖及分化形成胰岛素分泌细胞。各组在含有不同浓度葡萄糖的培养基中进行分化。采用免疫荧光染色法鉴定胰腺导管干细胞,光化学发光法检测分化出的胰岛素分泌细胞的胰岛素分泌量。 结果与结论：葡萄糖浓度为20.6,25.6,  30.6 mmol/L组细胞的刺激指数高于其他组(P < 0.05),但这3组两两比较差异无显著性意义(P > 0.05)。葡萄糖浓度为15.6,20.6,25.6 mmol/L组细胞胰岛素分泌量高于其他组(P < 0.05),但这3组两两比较差异无显著性意义(P > 0.05)。提示胰腺导管干细胞分化为胰岛素分泌细胞实验中,当分化时培养液所含葡萄糖浓度为20.6~25.6 mmol/L时,所得细胞胰岛素分泌能力最强。     

Urotensin Ⅱ诱导HepG2细胞胰岛素抵抗模型的建立及机制探讨

<正>目的:采用Urotensin Ⅱ(UⅡ)体外诱导法建立HepG 2细胞胰岛素抵抗模型,并探讨其机制。方法:采用含不同UⅡ浓度的DMEM培养基培养HepG 2细胞,最后半小时与1×10-7mol/L insulin共孵育,葡萄糖-己糖激酶法检测不同时点培养基中葡萄糖含量,以HepG 2细胞蛋白进行定量,确定胰岛素抵抗最佳作用浓度及时间;多功能酶标仪检测UⅡ诱导hepG 2细胞内活性     

莱菔硫烷诱导胰岛系INS-1细胞血红素氧合酶1的表达****◆○

背景：莱菔硫烷可用于氧化应激相关疾病的治疗,血红素氧合酶是一种催化血红素降解的应激蛋白,已经成为预防氧化攻击的首选研究靶标之一。目的：观察核因子E2相关因子2激动剂莱菔硫烷对大鼠胰岛细胞系INS-1细胞血红素氧合酶1蛋白表达作用及细胞保护机制。方法：体外培养INS-1细胞,先用3 μmol/L莱菔硫烷进行干预培养3 h,再分别加入不同的胰岛素抵抗诱导剂葡萄糖氧化酶、地塞米松和葡萄糖胺刺激建立胰岛素抵抗细胞模型。结果与结论：3 μmol/L莱菔硫烷处理INS-1细胞中血红素氧合酶1的表达增加(P < 0.05),其效应在4 h后达到高峰(P < 0.05)。3 μmol/L莱菔硫烷的预处理可逆转由胰岛素抵抗诱导剂导致的血红素氧合酶1表达下调(P < 0.05)。而且在葡萄糖胺处理的INS-1细胞中,莱菔硫烷对血红素氧合酶1的表达改善与磷酸化PKB的表达上调具有正相关性(P < 0.05, r = 0.23)。结果证实,莱菔硫烷可能通过诱导核因子E2相关因子2介导的血红素氧合酶1表达,增强胰岛细胞抗氧化防御功能和抗损伤信号系统,从而达到拮抗胰岛素抵抗诱导剂对胰岛细胞的损伤作用。     

前列腺素E1对人类脂肪源性基质细胞体外增殖的影响

背景：近年来脂肪源性基质细胞成为研究与应用的热点之一,但前列腺素E1对脂肪源性基质细胞增殖的作用影响尚未见报道。 目的：观察前列腺素E1对人类脂肪源性基质细胞体外增殖的影响。 方法：从人正常脂肪组织分离培养脂肪源性基质细胞,用5种不同浓度的前列腺素E1处理培养的细胞,其中3×10^-6,6×10^-6,9×10^-6,12×10^-6 mol/L作为实验组,0×10^-6 mol/L作为对照组,观察经不同浓度前列腺素E1处理后细胞的增殖情况。 结果与结论：不同浓度实验组的细胞数量与对照组相比均显著升高,差异有显著性意义(P < 0.05),各实验组内相比,细胞数量增殖无显著性差异(P  > 0.05)。提示前列腺素E1具有促进脂肪源性基质细胞的增殖作用,且前列腺素E1的影响效果无剂量依赖性。     

甲鱼油对胰岛素抵抗细胞模型胰岛素敏感性和葡萄糖代谢的影响

采用高浓度胰岛素诱导建立胰岛素抵抗(IR)HepG2细胞模型,评价甲鱼油对IR细胞模型胰岛素敏感性和葡萄糖代谢的影响.利用甲鱼油处理IR细胞模型,通过葡萄糖氧化酶及3H-D-葡萄糖参入实验分别检测其葡萄糖消耗和3H-D-葡萄糖参入率.结果表明,HepG2细胞模型的葡萄糖参入率在各种浓度的胰岛素刺激下均低于对照细胞(P＜0.05),并且HepG2细胞模型与对照细胞的3H-D-葡萄糖参入率随着胰岛素浓度的升高而升高,但前者升高的幅度低于后者.MTT(甲基噻唑基四唑)结果显示甲鱼油均具有促进对照细胞和IR细胞模型增殖作用.与对照组细胞相比,采用不同浓度胰岛素诱导的模型细胞经甲鱼油处理后其葡萄糖摄取和消耗显著增加(P＜0.05).甲鱼油明显增加该IR细胞模型的葡萄糖消耗和3H-D-葡萄糖参入率,提高IR细胞模型的胰岛素敏感性,改善其糖代谢.     

葛根素抑制1，25-二羟基维生素D3促破骨细胞骨吸收功能的最佳剂量

背景：近年来国内外学者对葛根素的骨形成研究较多,但对于葛根素抑制1,25-(OH)₂D₃促破骨细胞骨吸收功能尚未见报道。 目的：观察不同浓度葛根素在体外对1,25-(OH)₂D₃促破骨细胞骨吸收功能的抑制作用。 方法：收集3周龄小鼠骨髓细胞并接种到24孔培养板中。实验组培养液为1,25-(OH)₂D₃ +α-MEM完全培养基+不同质量浓度的葛根素,对照组不加葛根素。 结果与结论：不同浓度组葛根素处理的培养体系均诱导出破骨样细胞且形态差异不明显;除100 μg/L葛根素组在第3天时碱性磷酸酶表达显著增高外,各组之间培养液上清中碱性磷酸酶水平无显著性差异(P > 0.05);10 μg/L葛根素组培养液上清Ca2+水平在第3,5,12天时较对照组显著降低(P < 0.05~0.01),50 μg/L葛根素组培养液上清Ca²⁺水平在第12天时较对照组显著降低(P < 0.01),100 μg/L葛根素组培养液上清Ca²⁺水平在所有检测点较10 μg/L葛根素组显著上升(P < 0.05)。提示葛根素最佳抑制剂量在10~50 μg/L之间。     

乙烯雌酚可诱导骨髓基质干细胞向成骨细胞分化

背景：关于雌激素对骨髓基质干细胞作用的报道尚不多。 目的：观察乙烯雌酚对兔骨髓基质干细胞成骨分化的影响。 方法：体外培养骨髓基质干细胞,用0,10^-7,10^-6,10^-5 mol/L浓度乙烯雌酚干预,并设地塞米松10^-8 mol/L、β-甘油磷酸钠10 mmol/L、维生素C 50 mg/L为阳性对照。 结果与结论：乙烯雌酚干预培养24,48,72 h,10^-6 mol/L组显著促进了骨髓基质干细胞增殖(P < 0.01)。干预48 h 10^-5 mol/L组显著抑制细胞增殖,干预72 h 10^-7 mol/L组显著抑制细胞增殖(P < 0.01)。10^-7 mol/L组乙烯雌酚干预25 d后开始出现钙化结节;10^-7,10^-6 mol/L组干预14,21 d碱性磷酸酶活性显著增加。证实乙烯雌酚能促进兔骨髓基质干细胞的成骨分化。     

内分泌和化疗对乳腺肿瘤患者干细胞的影响

BACKGROUND:Tumor stem cells are the root of cancer recurrence and metastasis, so clinical researches should focus on the effects of different treatments on tumor stem cells. OBJECTIVE:To explore the effects of endocrine therapy and chemotherapy on stem cells in patients with breast cancer. METHODS:After recovery and cultivation of estrogen receptor-positive human breast cancer cell lines MCF-7, passage 3 cells in logarithmic phase were selected and divided into three groups containing control, estradiol and estradiol with tamoxifen groups. The estradiol group was divided into three subgroups: 10^-7, 10^-8 and 10^-9 mol/L estradiol was added into the medium, respectively; the estradiol with tamoxifen group was divided into three subgroups: 10^-7, 10^-8 and 10^-9 mol/L estradiol with 10^-6 mol/L tamoxifen were added into the medium, respectively. The same amount of absolute ethyl ethanol was added into the medium of control group. Fifteen female patients with late recurrence and metastasis of breast cancer received chemotherapy as recurrence and metastasis group. Another 15 healthy volunteers were selected as healthy control group. RESULTS AND CONCLUSION:The proportion of CD44⁺CD24^-/low cell subsets in the estradiol and estradiol with tamoxifen groups was significantly higher than that of the control group (P < 0.05), and the proportion of CD44⁺CD24^-/low cell subsets in the estradiol group was significantly higher than that of the estradiol with tamoxifen group at the same concentration (P < 0.05). The proportion of CD44+CD24-/low cell subsets had no significant differences among groups at 10 and 20 days of culture (P < 0.05). The proportion of CD44⁺CD24^-/low cell subsets significantly increased in MCF-7 cells after 24-hour intervention with different chemotherapy drugs. But only the proportion of CD44⁺CD24^-/low cell subsets in the paclitaxel and doxorubicin groups was significantly higher than that of the control group after 20-day intervention (P < 0.05). Besides, the proportion of CD44⁺CD24^-/low cell subsets in the peripheral blood of healthy volunteers was significantly lower than that of the recurrence and metastasis group (P < 0.05). Among 15 patients with late recurrence and metastatic of breast cancer, 9 had stable disease, 5 had partial remission, 1 had failed chemotherapy and cancer progression. Moreover, the proportion of CD45^-CD44⁺CD24^-/low cell subsets in the peripheral blood of patients sensitive for chemotherapy was significantly lower than that before treatment (P < 0.05). In conclusion, both endocrine therapy and chemotherapy exert a certain effect on the CD44⁺CD24^-/low cell subsets of breast cancer positive for estrogen receptor. Given that CD44⁺CD24^-/low cell subsets in MCF-7 cells resist chemotherapy drugs, the proportion of CD45^-CD44⁺CD24^-/low cells in the peripheral blood of patients sensitive for chemotherapy is decreased.     

成骨生长肽促进钛表面大鼠成骨细胞的增殖分化

背景：成骨生长肽具有促进多种基质细胞增殖活性,成骨活性,免疫原性低,自身调节极其敏感,提取制作工艺简单等众多优点。 目的：体外观察成骨生长肽对大鼠颅盖骨来源成骨细胞在钛金属表面增殖分化的影响。 方法：将体外培养的新生SD大鼠颅盖骨成骨细胞以5×10⁷ L^-1细胞浓度接种于6孔板中的纯钛试件表面,分别加入0(空白对照),10^-10,10^-9,10^-8,10^-7 mol/L的成骨生长肽,干预1,3,5,7,9 d后应用MTT法检测纯钛试件表面成骨细胞的增殖活性,应用酶联免疫法检测成骨细胞内碱性磷酸酶活性。 结果与结论：与空白对照组比较,各浓度成骨生长肽组纯钛试件表面成骨细胞增殖活跃(P < 0.05),且最佳作用浓度为10^-9 mol/L(P < 0.05);各浓度成骨生长肽组细胞内碱性磷酸酶活性增强(P < 0.05),且最佳作用浓度为10^-8 mol/L (P < 0.05)。表明成骨生长肽可促进钛片表面成骨细胞的增殖活性,增强细胞内碱性磷酸酶活性。     

CD44+CD24-/low乳腺癌干细胞活性与多药耐药的相关性

BACKGROUND:Tumor stem cells are found to be involved in the recurrence, metastasis and drug resistance of the tumor. OBJECTIVE:To explore the relationship between cell activity and multidrug resistance of CD44⁺CD24^-/low breast cancer stem cells. METHODS: CD44⁺CD24^-/low breast cancer stem cells sorted from multidrug resistant breast cancer cell line MCF-7/ADR were detected as percentage using flow cytometry. P-gp fluorescence intensity of the cell membrane and MDR mRNA expression in sorted cells and MCF-7/ADR were detected using flow cytometry and RT-PCR, respectively. RESULTS AND CONCLUSION:After sorting by flow cytometry, the proportion of CD44⁺CD24^-/low breast cancer stem cells was more than 90%, indicating that the sorted cells could meet the needs of the subsequent experiment. CD44⁺CD24^-/low cell subsets exhibited stronger ability to form microspheres than non- CD44⁺CD24^-/low cell subsets. The P-gp fluorescence intensity and MDR mRNA expression of CD44⁺CD24^-/low cells were significantly higher than those of MFC-7/ADR cell line (P < 0.05). These experimental findings suggest that CD44⁺CD24^-/low breast cancer stem cells sorted from MCF-7/ADR cell lines have a strong ability to form cell microspheres in vitro, and significantly raise the level of P-gp protein and MDR mRNA expression, which may be one of the causes of multidrug resistance.     

大麻素2型受体激活对钛颗粒诱导成骨细胞凋亡的干预作用

BACKGROUND: Impaired osteogenesis caused by osteoblast apoptosis is a main reason of aseptic loosening. Type-2 cannabinoid receptor activation can promote osteoblast proliferation and reduce apoptosis. OBJECTIVE: To investigate the inhibitory effect of HU-308 (type-2 cannabinoid receptor agonist) on the osteoblastic MC3T3-E1 cells co-cultured with titanium particles and its relative mechanism. METHODS: Mouse MC3T3-E1 cells were cultured in vitro and divided into five groups: cells were cultured in the normal medium as blank control group, cultured in the medium with 2.5 g/L titanium particles as titanium group, cultured in the medium containing 2.5 g/L titanium particles plus 10^-9, 10^-8 and 10-⁷ mol/L HU-308 as low-, medium- and high-concentration groups, respectively. Forty-eight hours later, cell proliferation, apoptosis, levels of reactive oxygen species and mitochondrial membrane potential as well as Caspase-3 activity were assessed. RESULTS AND CONCLUSION: Compared with the black control group, the cell proliferation activity and the level of mitochondrial membrane potential were significantly reduced and the apoptosis rate, the level of reactive oxygen species and Caspase-3 activity were significantly increased in the titanium group (P < 0.01 or P < 0.05). Compared with the titanium group, the cell proliferation activity and the level of mitochondrial membrane potential significantly increased in the high-concentration group, and the apoptosis rate and Caspase-3 activity significantly decreased in the medium- and high-concentration groups (P < 0.05). These findings demonstrate that HU-308 can effectively improve the impaired osteogenesis induced by titanium particles, probably through modulating reactive oxygen species and elevating mitochondrial membrane potential and Caspase-3 activity.      

骨髓间充质干细胞调节心脏移植大鼠的免疫功能

BACKGROUND:Heart transplantation is an effective method for treatment of end-stage heart failure, but immune rejection that seriously impact therapeutic effacicy is easy to occur after transplantation. OBJECTIVE:To investigate the regulatory effect of bone marrow mesenchymal stem cells on the immune function of rats undergoiong heart transplantation. METHODS:Twenty Lewis rats were enrolled as donors, and 20 Wistar rats as recipients. Heart transplantation models were established in the Wistar rats. These 20 model rats were randomized into cell transplantation and control group with 10 rats in each group. Forty-eight hours after heart transplantation, rats in the cell transplantation group were given bone marrow mesenchymal stem cell suspension (1 mL, 2×10⁸ cells/L) via the tail vein, while rats in the control group were given normal saline in the same dose. Then, the expression levels of serum interleukin-2, interleukin-10 and percentage of CD4⁺, CD8⁺, CD4⁺/CD8⁺, CD4⁺CD25^high, CD4⁺CD25^high Foxp3⁺ T cells in the venous blood were detected in the two groups at 7 days after cell transplantation. Additionally, rat myocardial tissues were taken and observed pathologically. RESULTS AND CONCLUSION:The survival time of the cell transplantation group was significantly longer than that of the control group (P < 0.05). The expression level of interleukin-2 showed no significant difference between the two groups (P > 0.05), but the level of interleukin-10 in the cell transplantation group was significantly higher than that in the control group (P < 0.05). Compared with the control group, the percentage of CD4⁺/CD8⁺, CD4⁺CD25^high, CD4⁺CD25^high Foxp3⁺ and CD4⁺ T cells was significantly higher, and the percentage of CD8⁺ T cells was significantly lower in the cell transplantation group (P < 0.05). Histopathological findings showed that there were a small amount of infiltrated lymphocytes in the cell transplantation group with the presence of slight bleeding and edema, and these inflammatory reactions were milder than those in the control group. These findings indicate that bone marrow mesenchymal stem cell transplantation can effectively reduce the rejection in rats undergoing heart transplantation.