本文關(guān)鍵詞： 乙型肝炎病毒 細(xì)胞凋亡 淋巴細(xì)胞 胸腺肽α1　出處：《南昌大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:研究HBV抗原對(duì)正常人淋巴細(xì)胞凋亡的影響及胸腺肽α1抑制HBV抗原誘導(dǎo)淋巴細(xì)胞凋亡的作用,并探討其意義。 方法: (一)淋巴細(xì)胞的分離: 取健康人靜脈血,肝素抗凝,用1640培養(yǎng)液對(duì)倍稀釋,緩緩加到淋巴細(xì)胞分離液上層,離心,吸取淋巴細(xì)胞,洗滌后用1640培養(yǎng)液配成2×10~6/ml的細(xì)胞懸液。 (二) HBV陽性血清(HBV(+)S)誘導(dǎo)淋巴細(xì)胞凋亡的實(shí)驗(yàn): 將淋巴細(xì)胞懸液分為四組:①胎牛血清(FCS)對(duì)照組:0.9ml淋巴細(xì)胞懸液+0.1ml FCS;②正常人血清(NHS)對(duì)照組:0.9ml淋巴細(xì)胞懸液+0.1ml NHS;③HBV(+)S實(shí)驗(yàn)組:0.9ml淋巴細(xì)胞懸液+ 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)陽性對(duì)照組:0.9ml淋巴細(xì)胞懸液+0.1ml FCS +DEX(終濃度為1×10~(-5)mol/L),每組設(shè)6個(gè)平行孔。將各組細(xì)胞接種于12孔細(xì)胞培養(yǎng)板,置37℃、5% CO2、飽和濕度的培養(yǎng)箱孵育,于培養(yǎng)不同時(shí)間收集培養(yǎng)細(xì)胞,分別用瑞氏染色法及原位缺口末端標(biāo)記法(TdT-mediated dUTP-biotin nick end labeling,TUNEL)觀察凋亡淋巴細(xì)胞形態(tài),計(jì)算凋亡率;流式細(xì)胞儀(FCM)碘化丙啶(PI)染色法分析亞二倍體峰,計(jì)算凋亡率。 同時(shí)設(shè)新鮮淋巴細(xì)胞對(duì)照組:取新分離淋巴細(xì)胞,不經(jīng)培養(yǎng),直接用上述方法檢測(cè)淋巴細(xì)胞凋亡情況。 (三)胸腺肽α1(Tα1)對(duì)淋巴細(xì)胞凋亡影響的實(shí)驗(yàn): 實(shí)驗(yàn)分組與上述相同,再于各組分別加入不同劑量的Tα1(終濃度分別為:0μg/ml、10μg/ml、50μg/ml與100μg/ml),每組設(shè)6個(gè)平行孔,其它步驟同前,于培養(yǎng)72h收集培養(yǎng)細(xì)胞,采用流式細(xì)胞儀檢測(cè)淋巴細(xì)胞凋亡率。 結(jié)果: (一)凋亡淋巴細(xì)胞的特征: 光學(xué)顯微鏡觀察凋亡細(xì)胞形態(tài)主要特征為:用瑞氏染色法光鏡下可見細(xì)胞核染色質(zhì)濃縮,邊聚及形成凋亡小體;TUNEL染色光鏡下可見凋亡淋巴細(xì)胞核染棕褐色;流式細(xì)胞術(shù)檢出凋亡特征性亞二倍體峰。 (二) HBV抗原誘導(dǎo)淋巴細(xì)胞凋亡的實(shí)驗(yàn)結(jié)果: HBV(+)S實(shí)驗(yàn)組凋亡率明顯高于NHS對(duì)照組及FCS對(duì)照組,差異具有顯著性(P0.01),提示HBV(+)S促進(jìn)淋巴細(xì)胞凋亡作用強(qiáng)于NHS,其凋亡率隨HBV DNA含量的升高而增加,低劑量組(HBV DNA含量為2.33×107 copy/ml)、中劑量組(HBV DNA含量為3.47×10~8 copy/ml)、高劑量組(HBV DNA含量為4.70×10~9 copy/ml)之間比較差異有顯著意義(P0.01)。其凋亡率與HBV DNA含量呈正相關(guān)(r=0.813, P0.01),并且細(xì)胞凋亡率的升高具有時(shí)間依賴性,未經(jīng)培養(yǎng)的淋巴細(xì)胞幾乎不出現(xiàn)凋亡,隨著培養(yǎng)時(shí)間的延長(zhǎng)凋亡率逐漸升高,培養(yǎng)24h、48h、72h組間比較,差異有顯著性(P0.01),淋巴細(xì)胞凋亡率與時(shí)間呈正相關(guān)(r=0.846, P0.01)。 (三) Tα1抑制HBV誘導(dǎo)淋巴細(xì)胞凋亡實(shí)驗(yàn)結(jié)果: 在FCS對(duì)照組、NHS對(duì)照組及HBV(+)S組分別加入中、高劑量Tα1(50μg/ml、100μg/ml)時(shí),與未加藥組(0μg/ml)比較,均顯示出明顯抑制淋巴細(xì)胞凋亡的作用(P0.01);但加入低劑量Tα1(10μg/ml)時(shí),僅有HBV(+)S組顯示出明顯抑制淋巴細(xì)胞凋亡的作用(P0.01),而NHS及FCS對(duì)照組與相應(yīng)未加藥組(0μg/ml)比較,未出現(xiàn)明顯抑制淋巴細(xì)胞凋亡的作用(P0.05)。Tα1不同劑量組間比較差異有顯著性意義(P0.01),Tα1對(duì)淋巴細(xì)胞凋亡抑制作用呈劑量依賴性,其藥物濃度與淋巴細(xì)胞凋亡率呈負(fù)相關(guān)(r=-0.683, P0.01)。 結(jié)論: ①本實(shí)驗(yàn)成功地建立了檢測(cè)淋巴細(xì)胞凋亡的實(shí)驗(yàn)方法,包括瑞氏染色法、TUNEL染色法、流式細(xì)胞儀術(shù)(PI染色)。 ②首次采用多種檢測(cè)細(xì)胞凋亡的方法較系統(tǒng)的觀察了HBV抗原體外對(duì)正常人淋巴細(xì)胞凋亡的影響,從形態(tài)學(xué)、細(xì)胞凋亡峰等多項(xiàng)指標(biāo)證實(shí)了HBV抗原在體外可誘導(dǎo)正常人淋巴細(xì)胞凋亡,并呈時(shí)間依賴性及劑量依賴性。 ③首次觀察了Tα1在體外對(duì)正常人淋巴細(xì)胞凋亡及HBV抗原誘導(dǎo)的淋巴細(xì)胞凋亡的影響,采用流式細(xì)胞儀檢測(cè)技術(shù)證實(shí)了Tα1在體外具有明顯抑制淋巴細(xì)胞凋亡的作用,并且對(duì)HBV抗原誘導(dǎo)的淋巴細(xì)胞凋亡作用更顯著。
[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.

【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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