淋巴细胞功能相关抗原-1基因敲除对实验性自身免疫性脑脊髓炎小鼠引流淋巴结T细胞活性的影响

目的探讨淋巴细胞功能相关抗原-1(lymphocyte function-associated antigen-1,LFA-1)基因敲除对实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)模型小鼠引流淋巴结T细胞活性的影响。方法采用髓鞘少突胶质细胞糖蛋白(myelin oligodendrocyte glycoprotein,MOG)35-55多肽诱导LFA-1a链CD11a基因敲除的C57BL/6小鼠及野生型C57BL/6小鼠,建立EAE小鼠模型,取MOG诱导后21 d EAE模型小鼠脊髓颈段切片,HE染色和LuxolFastBlue染色,观察小鼠脊髓组织学变化;于MOG诱导后7、14、21d处死小鼠,收集腹股沟引流淋巴结CD4+T淋巴细胞,加入终浓度为1μg/ml的MOG35-55多肽刺激48 h后,应用BrdU试剂盒检测细胞增殖情况;于MOG诱导后4、7、10、14、21 d处死小鼠,收集腹股沟引流淋巴结细胞,加入终浓度为1μg/ml的MOG35-55多肽刺激48 h后,进行细胞内细胞因子染色,观察分泌IFNγ和IL-17的T细胞比例。结果 MOG诱导的野生型小鼠脊髓出现明显淋巴细胞浸润及广泛的脱髓鞘变化,而LFA-1基因敲除小鼠无明显变化;在EAE疾病早期(7 d),LFA-1基因敲除可减少淋巴细胞向引流淋巴结聚集,降低MOG体外刺激T细胞增殖能力,分泌IFNγ及IL-17的T细胞比例也较野生型C57BL/6小鼠明显降低(P0.05);而在EAE疾病缓解期(21 d),LFA-1基因敲除小鼠淋巴细胞聚集及T细胞增殖能力与野生型C57BL/6小鼠相比无明显差异(P0.05),分泌IFNγ及IL-17的T细胞比例高于野生型C57BL/6小鼠(P0.05)。结论在EAE疾病发生早期,LFA-1可能起到了关键性的作用,LFA-1有可能成为自身免疫性疾病治疗的重要分子靶点。     

FoxO3a基因缺失致小鼠脾脏进行性慢性炎性反应的观察

目的观察FoxO3a基因缺失致小鼠脾脏进行性慢性炎性反应。方法分别于16周、24周和12个月称取FoxO3a基因敲除小鼠和对照小鼠体重;于16、24和38周处死各组小鼠,取脾脏,肉眼观察脾脏变化,并制备脾细胞悬液,进行细胞计数,同时取各组小鼠卵巢、肺及皮下等组织,观察其炎性变化。流式细胞术检测24周的FoxO3a基因敲除小鼠和对照小鼠脾细胞中T、B淋巴细胞和Mac-1+细胞数量及百分率;Real-time PCR检测脾细胞中炎性细胞因子S100A8和S100A9基因mRNA的表达水平。结果与对照小鼠相比,FoxO3a基因敲除小鼠脾脏明显肥大,且随年龄增加更加显著,卵巢、肺及皮下等组织内可见大量炎性细胞浸润,脾细胞数明显升高;基因敲除组老年小鼠的体重明显低于对照小鼠;FoxO3a基因敲除小鼠的脾细胞中T、B淋巴细胞数增加明显,但所占脾细胞的百分率无明显增加,Mac-1+细胞数和百分率均明显增加;S100A8和S100A9基因mRNA的表达水平明显高于对照小鼠。结论 FoxO3a基因缺失致小鼠脾脏进行性慢性炎性反应。     

维生素A对小鼠免疫调节功能的影响

目的探讨维生素A对小鼠免疫调节功能的影响。方法人工制备低维生素A、正常维生素A、高维生素A3种饲料,分别按常规饲养3组小鼠5周,应用微量荧光法测定血清维生素A浓度,三色流式细胞术测定CD4+T细胞内细胞因子IL-4和IFN-γ水平。结果3组小鼠血清维生素A浓度差异有显著意义,低维生素A组IFN-γ水平(28.60%±10.82%)明显高于正常对照组(20.13%±6.39%);高维生素A组IL-4水平(5.31%±2.04%)明显高于正常对照组(2.80%±1.37%),差异有非常显著意义(均P<0.01)。随着维生素A水平的提高,IFN-γ表达率降低,Th1免疫应答减弱,IL-4表达率增高,Th2免疫应答增强。结论维生素A对小鼠Th1/Th2免疫应答影响显著。     

血清中细胞因子在长效干扰素治疗慢性丙型肝炎前后的变化

目的观察聚乙二醇干扰素α-2α(PEG-IFN-α-2α)治疗慢性病毒性丙型肝炎前后免疫细胞因子的变化,探讨长效干扰素与细胞因子之间的关系,为长效干扰素治疗选择合适的适应证。方法双抗体夹心ELISA法对100例慢性病毒性丙型肝炎患者长效干扰素治疗前后IL-2、IL-4、IFN-γ、IL-10进行了检测,同时观察了T细胞亚群的变化。结果 100例丙肝患者,用长效干扰素治疗后,治疗有效41例,有效率达到41%,随访1～2年,10%复发。干扰素治疗前:慢性病毒性肝炎患者CD4+/CD8+比值、血清IL-2、IFN-γ水平降低,而IL-4、IL-10水平明显升高。治疗后:有效组CD4+/CD8+比值、血清IL-2、γ-IFN显著升高,IL-10明显下降治疗有效组IL-10显著低于治疗无效组(P<0.05),IL-4变化无统计学意义。而治疗无效组,治疗后仅IL-2较治疗前增高。CD4+/CD8+比值、血清IL-4、IL-10、IFN-γ较治疗前无明显变化。结论用长效干扰素治疗慢性病毒性丙型肝炎可以明显提高CD4+/CD8+比值,使T细胞活化,促进TH1因子的产生,抑制TH2细胞因子。T细胞亚群、TH1/Th2细胞因子的...     

非肥胖型糖尿病小鼠胸腺异位基因及MHC-Ⅱ类分子的表达

目的研究非肥胖型糖尿病(NOD)小鼠胸腺异位基因及MHC-Ⅱ类分子表达水平,探讨胸腺异位基因及MHC-Ⅱ类分子在中枢免疫耐受中的作用。方法对NOD小鼠用GA-PAP试剂盒检测血糖;尿糖试纸检测尿糖;ELISA间接法检测IAA的含量;光镜观察胰岛的组织学改变情况;MTT法检测胸腺T淋巴细胞功能;ELISA夹心法检测胸腺细胞IL-4、IFN-γ分泌水平;流式细胞术检测胸腺细胞MHC-Ⅱ类分子表达水平;RT-PCR法检测胸腺异位基因的表达水平。结果NOD小鼠血糖水平与正常BALB/c小鼠差异无显著意义,尿糖水平均为阴性;NOD小鼠自身抗体水平与正常BALB/c小鼠差异无显著意义;与对照组相比,NOD小鼠胰岛数量减少,分布稀疏,胰岛细胞数亦减少,同时有大量炎细胞浸润;NOD小鼠胸腺细胞对ConA刺激的应答水平与正常BALB/c小鼠差异无显著意义;NOD小鼠胸腺细胞IFN-γ的分泌与正常BALB/c小鼠差异无显著意义,而IL-4的分泌能力明显低于正常BALB/c小鼠;NOD小鼠胸腺内Insulin水平显著减少,而GAD67和PLP水平无显著变化;NOD小鼠MHC-Ⅱ类分子的表达水平明显下降。结论NOD小鼠在糖尿病发病前期存在着胸腺异位基因及MHC-Ⅱ类分子表达缺陷,表明胸腺异位基因及MHC-Ⅱ类分子在中枢免疫耐受中起着重要作用。     

CpG-ODN对伤寒Ty21a疫苗诱导的细胞免疫应答的作用

目的探讨CpG-ODN对伤寒Ty2la疫苗诱导的细胞免疫应答的作用。方法分别用含10、30及50μgCpG-ODN的伤寒Ty21a疫苗免疫小鼠,各剂量组分别于第0、14和28d灌胃免疫3次,末次免疫后1周,采集小鼠脾细胞。流式细胞仪检测脾细胞MHC-Ⅱ类分子和CD40分子的表达;培养脾细胞,用ELISA法检测脾细胞培养上清液中IFN-γ和IL-10的水平。结果不同剂量CpG-ODN的疫苗免疫组脾细胞CD40和MHC-Ⅱ类分子的表达水平均高于阳性对照组,且随着CpG-ODN剂量的增加,表达增强。加入不同剂量CpG-ODN的疫苗免疫组脾细胞培养上清液中IFN-γ和IL-10的水平均升高,且IL-10随着IFN-γ分泌水平的增加而降低。结论CpG-ODN可提高伤寒Ty21a疫苗诱导的Th1型免疫应答水平。     

糖基化磷脂酰肌醇锚定型人B7-1基因重组蛋白锚定自体肿瘤细胞膜提高抗肿瘤免疫反应的实验研究

目的研制抗肿瘤免疫治疗的新疫苗。方法用含目的基因的质粒pcDNA3-GPI-B7-1转染CHO/DHFR+细胞,提取膜蛋白GPI-B7-1,经Western blot鉴定后,用蛋白转化法锚定在肿瘤细胞膜上,免疫C57BL/6小鼠后,取小鼠脾细胞进行T细胞扩增和CTL功能检测,并检测细胞培养液和小鼠血清中IL-2、TNF-α和IFN-γ水平。结果用GPI-B7-1修饰的肿瘤细胞膜免疫小鼠后可诱发肿瘤特异性T细胞扩增和CTL活性增强,细胞培养液中细胞因子检测为阳性。结论该疫苗能激发小鼠的免疫功能,具有一定的保护小鼠免受肿瘤细胞侵袭的作用。     

口服CD226 DNA疫苗对小鼠免疫功能的影响

目的制备口服CD226 DNA疫苗,观察该疫苗对小鼠免疫功能的影响。方法以质粒CD226-PCR2.1-ToPo为模板,PCR扩增CD226基因,克隆至pcDNA3.1载体,构建真核表达质粒pcDNA3.1-CD226,利用脂质体LipofectamineTM2000瞬时转染CT-26细胞株,采用RT-PCR法、Western blot法、流式细胞术检测CD226基因在CT-26细胞中的表达。将质粒pcDNA3.1-CD226用脂质体Lipofectamine TM2000包裹制成CD226 DNA疫苗(100μg质粒/100μl脂质体),经灌胃免疫C57BL/6小鼠,实验分CD226疫苗组、pcDNA3.1组和生理盐水组,流式细胞术检测CD226在小鼠脾细胞中的表达;还原酶法检测NO分泌水平;乳酸脱氢酶释放法检测NK细胞杀伤活性;ELISA法检测脾细胞培养上清中细胞因子(IL-2、IL-4、IFNγ和TNF-α)分泌水平;Real-time PCR法检测肠黏膜组织内细胞因子表达水平。结果质粒pcDNA3.1-CD226经双酶切及测序鉴定,证明构建正确;质粒pcDNA3.1-CD226转染CT-26细胞的转染率为32.14%,转染的CT-26细胞检测到CD226蛋白表达。CD226疫苗组CD226 CD4+T细胞的绝对数、NK细胞杀伤活性、NO分泌水平、脾细胞培养上清中TNF-α、IFNγ和IL-2分泌水平、肠黏膜组织内TNF-α和IFNγ基因mRNA水平均明显高于pcDNA3.1组和生理盐水组(P0.05);而CD226疫苗组脾细胞培养上清中IL-4分泌水平及肠黏膜组织内IL-2和IL-4基因mRNA水平,与pcDNA3.1组和生理盐水组相比,差异无统计学意义(P0.05)。结论 CD226DNA疫苗经灌胃免疫,可诱导小鼠全身Th1型免疫应答增强和肠道局部部分Th1型免疫应答增强。     

内脂素在糖尿病模型小鼠糖脂代谢中的作用

目的探讨内脂素在糖尿病模型小鼠糖脂代谢中的作用。方法通过基因工程技术构建内脂素转基因和基因敲除小鼠,同时以野生小鼠作为对照,3组小鼠均以高脂高糖饮食饲养1个月后,于空腹12 h、进食30 min及1、2和3 h用血糖检测仪检测血糖水平;小鼠禁食10 h用生化分析仪检测甘油三酯、总胆固醇、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇的水平;Western blot法检测小鼠体内内脂素表达水平;相应试剂盒检测小鼠体内胰岛素受体及胰岛素水平。结果除空腹12 h组外,进食30 min及1、2和3 h转基因组小鼠血糖水平明显低于基因敲除组和野生组(P 0. 05),野生组小鼠血糖水平显著低于基因敲除组(P 0. 01);转基因组小鼠甘油三酯、总胆固醇、高密度脂蛋白胆固醇水平显著低于基因敲除组(P 0. 05);基因敲除组小鼠内脂素表达水平明显低于转基因组和野生组(P 0. 001),转基因组小鼠明显高于野生组(P 0. 001);转基因小鼠胰岛素受体水平显著高于基因敲除小鼠和野生型小鼠(P 0. 01),野生型组明显高于基因敲除组(P 0. 05),3组间胰岛素水平差异无统计学意义(P 0. 05)。结论内脂素通过与胰岛素非竞争性结合胰岛素受体而发挥降低糖尿病小鼠血糖作用,且在脂代谢中具有促进脂肪积累的功能。     

肠道病毒71型灭活疫苗的小鼠细胞免疫效果

目的评价肠道病毒71型(enterovirus 71,EV71)灭活疫苗诱导小鼠的细胞免疫效果。方法分别以不同剂量(1、2.5、5、10μg/ml,均含铝佐剂1 mg/ml)、不同剂型(含铝佐剂1 mg/ml或不含铝佐剂)、不同免疫剂次(单次免疫或加强免疫)EV71灭活疫苗经腹腔免疫小鼠,0.5 ml/只,均设铝佐剂对照组(仅注射铝佐剂1 mg/ml)。采用经典小鼠树突状细胞(dendritic cell,DC)培养方法制备正常小鼠DC,瑞氏染色法检测细胞形态,流式细胞术分析其表型及纯度;免疫磁珠分选法(magnetic activated cell sorting,MACS)分离小鼠脾脏CD4+、CD8+T淋巴细胞,流式细胞术检测细胞纯度。ELISPOT法检测各组免疫小鼠CD4+、CD8+T淋巴细胞分泌IL-2、IL-4、IFNγ的水平;细胞因子试剂盒检测小鼠淋巴细胞培养上清及小鼠血清中细胞因子的水平。结果 DC形态不规则,表面树枝状突起形态不同,细胞核较大且不规则;DC纯度为(81.39±9.24)%,可高水平表达MHⅡ(I-A/I-E)类分子,中度表达CD86和CD40。CD4+、CD8+T细胞纯度分别为95.27%和94.08%。随着疫苗免疫剂量的增加,CD4+T细胞分泌IL-2、IL-4、IFNγ及CD8+T细胞分泌IFNγ的SFC显著增加,5μg/ml疫苗组达最大值,且明显高于其他组(P均0.05);5μg/ml疫苗组淋巴细胞培养上清中IFNγ、IL-2、IL-4、IL-6、IL-10、IL-13、IL-5、TNF-α含量明显高于其他组(P0.05);1、2.5、5μg/ml疫苗组血清中IL-10含量明显高于铝佐剂对照组(P0.05),1μg/ml疫苗组明显低于2.5及5μg/ml疫苗组(P0.05)。含铝佐剂疫苗组CD4+T细胞分泌IL-2、IL-4、IFNγ及CD8+T细胞分泌IFNγ的SFC、淋巴细胞培养上清中IL-2、IFNγ、IL-4、IL-5、IL-6、IL-10、IL-13、TNF-α水平及血清中IL-10含量均明显高于无铝佐剂疫苗组(P均0.05)。加强免疫组CD4+T细胞分泌IL-2、IL-4、IFNγ及CD8+T细胞分泌IFNγ的SFC、淋巴细胞培养上清中IL-2、IFNγ、IL-4、IL-5、IL-6、IL-10、IL-13、TNF-α水平、血清中IL-10含量均明显高于单次免疫组(P均0.05)。结论 EV71灭活疫苗可诱导小鼠产生特异性细胞免疫反应,本实验为其进一步人体临床试验研究奠定了基础。     

Kurarinone Attenuates Collagen-Induced Arthritis in Mice by Inhibiting Th1/Th17 Cell Responses and Oxidative Stress

Kurarinone is a flavanone, extracted from Sophora flavescens Aiton, with multiple biological effects. Here, we determine the therapeutic potential of kurarinone and elucidate the interplay between kurarinone and the autoimmune disease rheumatoid arthritis (RA). Arthritis was recapitulated by induction of bovine collagen II (CII) in DBA/1 mice as a collagen-induced arthritis (CIA) model. After the establishment of the CIA, kurarinone was given orally from day 21 to 42 (100 mg/kg/day) followed by determination of the severity based on a symptom scoring scale and with histopathology. Levels of cytokines, anti-CII antibodies, and the proliferation and lineages of T cells from the draining lymph nodes were measured using ELISA and flow cytometry, respectively. The expressional changes, including STAT1, STAT3, Nrf2, KEAP-1, and heme oxygenase-1 (HO-1) changes in the paw tissues, were evaluated by Western blot assay. Oxidative stress featured with malondiadehyde (MDA) and hydrogen peroxide (H₂O₂) activities in paw tissues were also evaluated. Results showed that kurarinone treatment reduced arthritis severity of CIA mice, as well as their levels of proinflammatory cytokines, TNF-α, IL-6, IFN-γ, and IL-17A, in the serum and paw tissues. T cell proliferation was also reduced by kurarinone even under the stimulation of CII and anti-CD3 antibody. In addition, kurarinone reduced STAT1 and STAT3 phosphorylation and the proportions of Th1 and Th17 cells in lymph nodes. Moreover, kurarinone suppressed the production of MDA and H₂O_2. All while promoting enzymatic activities of key antioxidant enzymes, SOD and GSH-Px. In the paw tissues, upregulation of Nrf-2 and HO-1, and downregulation of KEAP-1 were observed. Overall, kurarinone showed an anti-inflammatory effect by inhibiting Th1 and Th17 cell differentiation and an antioxidant effect exerted in part through activating the Nrf-2/KEAP-1 pathway. These beneficial effects in CIA mice contributed to the amelioration of their arthritis, indicating that kurarinone might be an adjunct treatment option for rheumatoid arthritis.     

口蹄疫病毒多价DNA疫苗的构建及其免疫原性

目的构建口蹄疫病毒(FMDV)多价DNA疫苗,并检测其免疫原性。方法以复合多表位表达盒OAAT及AsiaⅠ型FMDV的P1-2A-3C基因为基础,构建FMDV多价DNA疫苗pIRES-OAAT-P1-2A-3C,并用间接免疫荧光(IFA)方法检测目的蛋白在HeLa细胞中的表达。进一步进行小鼠免疫试验,并应用ELISA法检测小鼠血清抗体,ELISPOT检测小鼠脾淋巴单细胞IFN-γ的分泌水平,流式细胞术检测脾T淋巴细胞亚群数量,淋巴细胞转化试验检测特异性淋巴细胞增殖水平。结果所构建的FMDV多价DNA疫苗在HeLa细胞中获得了正确表达。免疫小鼠后,血清特异性抗体水平、脾淋巴单细胞IFN-γ的分泌、脾T淋巴细胞亚群CD4+和CD8+的数量及特异性淋巴细胞增殖水平均显著提高。结论已成功构建了FMDV多价DNA疫苗,并诱导小鼠产生了特异性的细胞免疫和体液免疫应答。     

The PI-3-Kinase P110α Catalytic Subunit of T Lymphocytes Modulates Collagen-Induced Arthritis

The phosphatidylinositol 3-kinase (PI3K) family of enzymes plays a determinant role in inflammation and autoimmune responses. However, the implication of the different isoforms of catalytic subunits in these processes is not clear. Rheumatoid arthritis (RA) is a chronic, systemic autoimmune inflammatory disease that entails innate and adaptive immune response elements in which PI3K is a potential hub for immune modulation. In a mouse transgenic model with T-cell-specific deletion of p110α catalytic chain (p110α^−/−ΔT), we show the modulation of collagen-induced arthritis (CIA) by this isoform of PI3K. In established arthritis, p110α^−/−ΔT mice show decreased prevalence of illness than their control siblings, higher IgG1 titers and lower levels of IL-6 in serum, together with decreased ex vivo Collagen II (CII)-induced proliferation, IL-17A secretion and proportion of naive T cells in the lymph nodes. In a pre-arthritis phase, at 13 days post-Ag, T-cell-specific deletion of p110α chain induced an increased, less pathogenic IgG1/IgG2a antibodies ratio; changes in the fraction of naive and effector CD4⁺ subpopulations; and an increased number of CXCR5⁺ T cells in the draining lymph nodes of the p110α^−/−ΔT mice. Strikingly, T-cell blasts in vitro obtained from non-immunized p110α^−/−ΔT mice showed an increased expression of CXCR5, CD44 and ICOS surface markers and defective ICOS-induced signaling towards Akt phosphorylation. These results, plus the accumulation of cells in the lymph nodes in the early phase of the process, could explain the diminished illness incidence and prevalence in the p110α^−/−ΔT mice and suggests a modulation of CIA by the p110α catalytic chain of PI3K, opening new avenues of intervention in T-cell-directed therapies to autoimmune diseases.     

重组人IL-12真核表达载体的基因佐剂功能

目的探讨重组人IL-12真核表达载体(pcDNA6-p70)对核酸疫苗(HCMV pp65基因重组腺病毒,Adeno-pp65)的基因佐剂功能。方法pcDNA6-p70与Adeno-pp65共免疫BALB/c小鼠,每2周1次,共4次,同时设立Adeno-pp65对照组及生理盐水对照组。于初次免疫后第6周末尾静脉取血,第9周处死小鼠,分离脾细胞,分别检测细胞内细胞因子水平、CTL杀伤活性、特异性淋巴细胞增生和IFN-γ水平,并观察pcDNA6-p70对小鼠的安全性。结果共免疫组HCMV特异性CD4/IFN-γ、CD8/IFN-γ双标记阳性细胞的百分率均高于Adeno-pp65组及生理盐水对照组,差异均有显著意义;其CTL杀伤活性、HCMV特异性淋巴细胞增生水平及脾淋巴细胞特异性IFN-γ释放水平均高于Adeno-pp65组和生理盐水对照组,差异均有显著意义。pcDNA6-p70肌肉注射昆明小鼠,其体温、体重及一般情况与对照组差异均无显著意义。结论pcDNA6-p70与Adeno-pp65共免疫可提高核酸疫苗的免疫效果,且具有较好的安全性。     

Berberine Delays Onset of Collagen-Induced Arthritis through T Cell Suppression

There is evidence that berberine (BBR), a clinically relevant plant compound, ameliorates clinically apparent collagen-induced arthritis (CIA) in vivo. However, to date, there are no studies involving the use of BBR which explore its prophylactic potential in this model of rheumatoid arthritis (RA). The aim of this study was to determine if prophylactic BBR use during the preclinical phase of collagen-induced arthritis would delay arthritic symptom onset, and to characterize the cellular mechanism underlying such an effect. DBA/1J mice were injected with an emulsion of bovine type II collagen (CII) and complete Freund’s adjuvant (day 0) and a booster injection of CII in incomplete Freund’s adjuvant (day 18) to induce arthritis. Mice were then given i.p. injections of 1 mg/kg/day of BBR or PBS (vehicle with 0.01% DMSO) from days 0 to 28, were left untreated (CIA control), or were in a non-arthritic control group (n = 15 per group). Incidence of arthritis in BBR-treated mice was 50%, compared to 90% in both the CIA and PBS controls. Populations of B and T cells from the spleens and draining lymph nodes of mice were examined on day 14 (n = 5 per group) and day 28 (n = 10 per group). BBR-treated mice had significantly reduced populations of CD4⁺T_h and CD4⁺CXCR5⁺ T_fh cells, and an increased proportion of Foxp3⁺ T_reg at days 14 and 28, as well as reduced expression of co-stimulatory molecules CD28 and CD154 at both endpoints. The effect seen on T cell populations and co-stimulatory molecule expression in BBR-treated mice was not mirrored in CD19⁺ B cells. Additionally, BBR-treated mice experienced reduced anti-CII IgG2a and anti-CII total IgG serum concentrations. These results indicate a potential role for BBR as a prophylactic supplement for RA, and that its effect may be mediated specifically through T cell suppression. However, the cellular effector involved raises concern for BBR prophylactic use in the context of vaccine efficacy and other primary adaptive immune responses.     

血脂康对新生大鼠心肌成纤维细胞增殖及胶原合成的影响

目的观察血脂康对血管紧张素Ⅱ(AngⅡ)诱导的新生大鼠心肌成纤维细胞(CFs)增殖及胶原合成的影响,并探讨可能的分子机制。方法采用胰酶消化法分离培养Sprague-Dawley大鼠心肌成纤维细胞,建立AngⅡ诱导CFs增殖的模型,以MTT比色法和流式细胞仪检测细胞周期分析法观察血脂康对CFs数目和细胞周期的影响。AngⅡ及不同浓度血脂康作用48 h后,用天狼星红染色法检测培养上清中胶原的含量;ELISA法检测细胞培养上清液中TGF-β1蛋白表达;RT-PCR法检测胶原和TGF-β1 mRNA表达。结果AngⅡ对CFs增殖有明显促进作用,血脂康可明显抑制AngⅡ诱导的CFs增殖,且呈剂量依赖性;血脂康可增加G0/G1期细胞百分率,降低S、G2/M期细胞百分率,降低胶原含量、TGF-β1蛋白及胶原和TGF-β1 mRNA的表达。结论血脂康能抑制AngⅡ诱导的CFs增殖和胶原的产生,其作用可能是通过抑制TGF-β1表达实现的。