HBV抗原诱导正常人淋巴细胞凋亡及胸腺肽α1对其凋亡抑制作用的研究

发布时间：2018-01-20 03:32

本文关键词： 乙型肝炎病毒细胞凋亡淋巴细胞胸腺肽α1　出处：《南昌大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:研究HBV抗原对正常人淋巴细胞凋亡的影响及胸腺肽α1抑制HBV抗原诱导淋巴细胞凋亡的作用,并探讨其意义。方法: (一)淋巴细胞的分离: 取健康人静脉血,肝素抗凝,用1640培养液对倍稀释,缓缓加到淋巴细胞分离液上层,离心,吸取淋巴细胞,洗涤后用1640培养液配成2×10~6/ml的细胞悬液。 (二) HBV阳性血清(HBV(+)S)诱导淋巴细胞凋亡的实验: 将淋巴细胞悬液分为四组:①胎牛血清(FCS)对照组:0.9ml淋巴细胞悬液+0.1ml FCS;②正常人血清(NHS)对照组:0.9ml淋巴细胞悬液+0.1ml NHS;③HBV(+)S实验组:0.9ml淋巴细胞悬液+ 0.1ml HBV(+)S(不同HBV DNA含量:2.33×10~7 copy/ml、3.47×10~8 copy/ml、4.70×10~9 copy/ml);④地塞米松(DEX)阳性对照组:0.9ml淋巴细胞悬液+0.1ml FCS +DEX(终浓度为1×10~(-5)mol/L),每组设6个平行孔。将各组细胞接种于12孔细胞培养板,置37℃、5% CO2、饱和湿度的培养箱孵育,于培养不同时间收集培养细胞,分别用瑞氏染色法及原位缺口末端标记法(TdT-mediated dUTP-biotin nick end labeling,TUNEL)观察凋亡淋巴细胞形态,计算凋亡率;流式细胞仪(FCM)碘化丙啶(PI)染色法分析亚二倍体峰,计算凋亡率。同时设新鲜淋巴细胞对照组:取新分离淋巴细胞,不经培养,直接用上述方法检测淋巴细胞凋亡情况。 (三)胸腺肽α1(Tα1)对淋巴细胞凋亡影响的实验: 实验分组与上述相同,再于各组分别加入不同剂量的Tα1(终浓度分别为:0μg/ml、10μg/ml、50μg/ml与100μg/ml),每组设6个平行孔,其它步骤同前,于培养72h收集培养细胞,采用流式细胞仪检测淋巴细胞凋亡率。结果: (一)凋亡淋巴细胞的特征: 光学显微镜观察凋亡细胞形态主要特征为:用瑞氏染色法光镜下可见细胞核染色质浓缩,边聚及形成凋亡小体;TUNEL染色光镜下可见凋亡淋巴细胞核染棕褐色;流式细胞术检出凋亡特征性亚二倍体峰。 (二) HBV抗原诱导淋巴细胞凋亡的实验结果: HBV(+)S实验组凋亡率明显高于NHS对照组及FCS对照组,差异具有显著性(P0.01),提示HBV(+)S促进淋巴细胞凋亡作用强于NHS,其凋亡率随HBV DNA含量的升高而增加,低剂量组(HBV DNA含量为2.33×107 copy/ml)、中剂量组(HBV DNA含量为3.47×10~8 copy/ml)、高剂量组(HBV DNA含量为4.70×10~9 copy/ml)之间比较差异有显著意义(P0.01)。其凋亡率与HBV DNA含量呈正相关(r=0.813, P0.01),并且细胞凋亡率的升高具有时间依赖性,未经培养的淋巴细胞几乎不出现凋亡,随着培养时间的延长凋亡率逐渐升高,培养24h、48h、72h组间比较,差异有显著性(P0.01),淋巴细胞凋亡率与时间呈正相关(r=0.846, P0.01)。 (三) Tα1抑制HBV诱导淋巴细胞凋亡实验结果: 在FCS对照组、NHS对照组及HBV(+)S组分别加入中、高剂量Tα1(50μg/ml、100μg/ml)时,与未加药组(0μg/ml)比较,均显示出明显抑制淋巴细胞凋亡的作用(P0.01);但加入低剂量Tα1(10μg/ml)时,仅有HBV(+)S组显示出明显抑制淋巴细胞凋亡的作用(P0.01),而NHS及FCS对照组与相应未加药组(0μg/ml)比较,未出现明显抑制淋巴细胞凋亡的作用(P0.05)。Tα1不同剂量组间比较差异有显著性意义(P0.01),Tα1对淋巴细胞凋亡抑制作用呈剂量依赖性,其药物浓度与淋巴细胞凋亡率呈负相关(r=-0.683, P0.01)。结论: ①本实验成功地建立了检测淋巴细胞凋亡的实验方法,包括瑞氏染色法、TUNEL染色法、流式细胞仪术(PI染色)。 ②首次采用多种检测细胞凋亡的方法较系统的观察了HBV抗原体外对正常人淋巴细胞凋亡的影响,从形态学、细胞凋亡峰等多项指标证实了HBV抗原在体外可诱导正常人淋巴细胞凋亡,并呈时间依赖性及剂量依赖性。 ③首次观察了Tα1在体外对正常人淋巴细胞凋亡及HBV抗原诱导的淋巴细胞凋亡的影响,采用流式细胞仪检测技术证实了Tα1在体外具有明显抑制淋巴细胞凋亡的作用,并且对HBV抗原诱导的淋巴细胞凋亡作用更显著。
[Abstract]:Objective: To study the effect of HBV antigen on the apoptosis of normal human lymphocytes and the effect of thymosin alpha 1 on the inhibition of HBV antigen induced lymphocyte apoptosis and to explore its significance.
Method:
(I) the separation of lymphocytes:
The venous blood of healthy people and heparin anticoagulation were taken, diluted with 1640 culture medium, slowly added to the upper layer of lymphocyte separation fluid, centrifugated, lymphocytes were collected, and then washed with 1640 culture medium to form 2 x 10~6/ml cell suspension.
(two) the experiment of lymphocyte apoptosis induced by HBV positive serum (HBV (+) S):
The cell suspension was divided into four groups: fetal bovine serum (FCS) control group: 0.9ml lymphocyte suspension +0.1ml FCS; normal human serum (NHS) control group: 0.9ml lymphocyte suspension of +0.1ml NHS; the HBV (+) S group: 0.9ml lymphocyte suspension + 0.1ml HBV (S (+) different HBV DNA content: 2.33 x 10~7 copy/ml 3.47 * 10~8 4.70 * 10~9 copy/ml, copy/ml); the dexamethasone (DEX) positive control group: 0.9ml +0.1ml FCS +DEX (lymphocyte suspension at the concentration of 1 * 10~ (-5) mol/L), each with 6 parallel holes. The cells were inoculated in 12 holes cell culture plate, 37 DEG C, 5% CO2, saturated humidity incubator incubation incubation in different cultivation time collecting cells respectively by Wright staining and TUNEL (TdT-mediated dUTP-biotin nick end labeling, TUNEL) to observe the apoptosis of lymphocyte morphology, apoptosis rate was calculated by flow cytometry (FCM; propidium iodide (PI)) The staining method was used to analyze the subdiploid peak and calculate the apoptosis rate.
At the same time, the fresh lymphocyte control group was set up, and the lymphocyte apoptosis was detected directly by the new isolated lymphocyte and without culture.
(three) the experiment of the effect of thymosin alpha 1 (T alpha 1) on lymphocyte apoptosis:
The experimental group with the same, and in each group with different doses of T alpha 1 (final concentrations: 0 g/ml, 10 g/ml, 50 g/ml and 100 g/ml), each with 6 parallel holes, with other steps, in cultured 72h cells apoptosis by collection, flow cytometry the detection rate of lymphocytes.
Result:
(I) characteristics of apoptotic lymphocytes:
The main features of optical microscope: the morphology of apoptotic cells with Wright staining under light microscope, chromatin condensation, margination and apoptotic body formation; TUNEL staining under light microscope, apoptotic lymphocyte nuclear staining in brown; flow cytometry detection feature of apoptosis was two times as much as the sub peak.
(two) the experimental results of lymphocyte apoptosis induced by HBV antigen:
HBV (+) S group apoptosis rate was obviously higher than that of NHS control group and FCS control group, the difference was significant (P0.01), HBV (+) S promotes apoptosis of lymphocytes was stronger than NHS, the apoptosis rate of HBV increased with the DNA content increasing, the low dose group (HBV 2.33 * 107 DNA content copy/ml), middle dose group (HBV 3.47 * 10~8 copy/ml DNA in the high dose group (HBV), the content of DNA is 4.70 * 10~9 copy/ml) with a significant difference (P0.01). The apoptosis rate of HBV was positively related with the content of DNA (r=0.813, P0.01), and the cell apoptosis rate increased with time dependence no, there is almost no cultured lymphocyte apoptosis, with prolonged incubation time, apoptosis rate increased gradually, 24h culture, 48h, 72h group, there was significant difference (P0.01), lymphocyte apoptosis rate and time was positively correlated (r=0.846, P0.01).
(three) T alpha 1 inhibits the experimental results of lymphocyte apoptosis induced by HBV:
In the FCS control group, NHS control group and HBV (+) S groups were added in high dose T alpha 1 (50 g/ml, 100 g/ml), and no drug group (0 g/ml), showed a significant inhibition of lymphocyte apoptosis (P0.01); but with a low dose of T alpha 1 (10 g/ml), only HBV (+) S group showed significant inhibition of lymphocyte apoptosis (P0.01), NHS and FCS in control group and no drug group (0 g/ml), there was no obvious inhibition of lymphocyte apoptosis (P0.05) with significant.T alpha 1 different doses of difference between the two groups (P0.01, T) alpha 1 inhibitory effect on lymphocyte apoptosis in a dose-dependent manner, the drug concentration and lymphocyte apoptosis rate was negatively correlated (r=-0.683, P0.01).
Conclusion:
(1) the experimental methods for detecting lymphocyte apoptosis were successfully established in this experiment, including Rayleigh staining, TUNEL staining, and flow cytometry (PI staining).
For the first time by using the method of detecting apoptosis of various systematic effects, HBV antigen in vitro on apoptosis of lymphocytes from normal morphology, apoptosis peak index confirmed that HBV antigen can induce apoptosis of normal human lymphocytes in vitro, and showed a time dependent and dose dependent manner.
For the first time, the effect of T on normal human alpha 1 induced lymphocyte apoptosis and HBV antigen in vitro lymphocyte apoptosis, confirmed T alpha 1 in vitro inhibited the lymphocyte apoptosis by flow cytometry technique, and the role of HBV antigen induced lymphocyte apoptosis is more significant.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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