【作者】 白山岭；

【导师】 赵映前；

【作者基本信息】 湖北中医药大学 ， 中医临床基础， 2014， 博士

【摘要】 目的：基于肝癌的发病机制与细胞增殖和凋亡的失衡密切相关，以P53、Bax、Bcl-2与肝癌细胞凋亡明确相关的基因为切入点，进行相关研究，观察牛黄参（NHS）含药血清体外诱导人肝癌细株HepG2细胞凋亡的作用，初步探讨其作用机制，为牛黄参胶囊的研究与应用提供理论与实验依据以及为临床治疗原发型肝癌开辟一条新思路。方法：采用药物血清学和细胞药理学方法，对比观察（阴性无药物血清对照、阳性药CTX对照）NHS含药血清对HepG2细胞凋亡及凋亡相关调控基因Bax，Bc1-2及抑癌基因P53的影响。主要观察指标及检测方法如下：1．含药血清的制备：40只SD大鼠，随机分为空白对照组（8只）、CTX含药血清组（8只）和牛黄参含药血清组（24只）。以成人临床一日常用量为含药血清组的有效剂量，按人和大鼠体表面积折算的等效剂量比值换算出大鼠的一日用药剂量，并设为含药血清组终剂量。空白对照组：灌服等效生理盐水每天两次，每次5mL。含药血清组：CTX水溶液（25.2mg/kg）每天灌胃两次，每次5mL；牛黄参煎液（1.07g/kg）每天灌胃两次，每次5mL；重复给药连续七天，末次给药45分钟后采取心脏穿刺采血法取血，将采集的血液在室温中静置4h，3000rpm，离心15min，收集血清，将收集的血清在56℃水浴30min进行灭活，而后在超净台内用0.22pm微孔滤膜过滤除菌，将血清移至干净试管中，置-20℃保存备用，需要时用培养液配制成所需浓度的含药血清。2．以CCK-8法检查五个药物血清浓度对IIepC2细胞增殖的抑制作用，从中找出NHS的最佳有效药物血清浓度。3．HepG2细胞NHS含药血清给药48h后，AO/EB和DAPI荧光染色，荧光显微镜下观察细胞形态学变化。4．流式细胞术AnnexinV-FITC/PI双染色法检测NHS含药血清对HepG2细胞周期和凋亡百分率影响。5．蛋白质印迹法（Western Blot）检测NHS含药血清对HepG2细胞的P53、Bcl-2基因蛋白表达的影响。6．免疫组化方法检测NHS含药血清三个有效药物浓度对HepG2细胞Bcl-2, Bax及细胞核增殖性抗原(PCNA)表达的影响。7．Real time RT-PCR检测含药血清处理前、后的HepG2细胞中Bcl-2和Bax mRNA的相对表达。结果：1. CTX组及牛黄参各浓度组细胞增殖受到明显抑制，且随着浓度增加和作用时间延长，HepG2细胞的增殖数目和速度下降，牛黄参各浓度组与空白对照组比较，差异有统计学意义(P＜0.05，P＜0.01)。2.通过荧光显微镜观察到典型的细胞形态学改变：含药血清组部分的细胞核呈现折缝样、波纹状，核染色质着桔红色，部分染色质出现浓缩状态；CTX含药血清组、20%NHS含药血清组可见大量不同程度的坏死和凋亡细胞，其细胞体积变小，核膜消失，胞质内空泡增多，细胞器较少，细胞核染色质固缩，形态不规则，可见核碎裂，产生凋亡小体，且培养时间越长，改变越显著。3．流式细胞仪分析HepG2细胞呈现出明显的亚二倍体凋亡峰，且细胞凋亡数量随着给药剂量的增加而增加。HepG2细胞在CTX含药血清、5%NHS含药血清、10%NHS含药血清和20%NHS含药血清给药48h后，凋亡细胞所占比例分别为30.13%、11.05%、19.76%和28.32%。细胞周期分析说明NHS含药血清组使该细胞生长阻止于G0/G1期。4．蛋白质印迹法(Western Blot)结果显示：与空白对照组相比，牛黄参含药血清作用HepG2细胞后，Bcl-2蛋白的表达量显著下降（P<0.05），P53蛋白的表达量显著上升（P<0.05），且两两组间相比有显著性差异（P<0.05）。5．免疫组化结果显示：随着NHS含药血清给药浓度的增加，PCNALI逐渐降低，表明NHS含药血清可抑制HepG2肿瘤细胞的增殖，且随着NHS含药血清浓度增加，能够明显降低HepG2细胞内Bcl-2蛋白的阳性表达率，Bax蛋白表达呈中强性表达，20%NHS、CTX组和10%NHS组阳性表达率分别为56.89%，58.33%，44.73%，与空白对照组比较，具有显著性差异(P<0.01，P<0.05)。6．实时荧光定量PCR检测各组细胞的CT值，分析各细胞凋亡相关因子mRNA相对表达水平显示,随着NHS含药血清浓度的增加Bax mRNA的表达水平增加，而Bcl-2mRNA的表达水平却降低。结论：1. NHS含药血清能在一定程度上抑制HepG2细胞的增殖。2. NHS含药血清具有诱导HepG2细胞凋亡的作用。3. NHS含药血清诱导HepG2细胞凋亡的作用机制之一可能是上调促凋亡基因Bax和P53的表达，下调Bcl-2的表达有关。更多还原

【Abstract】 Objective：Based on closely related to the pathogenesis of cell proliferation andapoptosis of liver imbalance to P53, Bax, Bcl-2and apoptosis inhepatocellular carcinoma clearly related genes as a starting point, conductresearch, observe NHS containing serum vitro differentiation of humanhepatoma cell line HepG2apoptosis, explore its mechanism of action,provide theoretical and experimental basis for the study and applicationof NHS capsules for the clinical treatment of primary hepatocellularcarcinoma has opened a new idea.Methods：The use of drugs and cellular pharmacology serological methods,comparative observation(negative serum control drug, the positive controldrug CTX) NHS containing serum on apoptosis of HepG2and regulationof apoptosis-related tomb because of Bax, Bc1-2and tumor suppressorgene P53impact. Main outcome measures and testing methods are asfollows:1.Preparation of serum containing:40SD rats were randomlydivided into control group(8),CTX-containing serum group(8) and NHSreference serum containing group(24). A daily dosage of adult clinical effective dose of the drug-containing serum group,dose equivalentconversion by human and rat body surface area ratio of the translation ofthe day dose in rats, and set the final dose of the drug-containing serumgroup.Control group: the equivalent saline gavage twice a day, every5mL.Containing serum group:CTX solution(25.2mg/kg) administered twicedaily, each5mL;NHS capsules decoction(1.07g/kg) orally twice a day,every5mL; repeated administration for seven consecutive days,45minutes after the last administration, blood taken by cardiac puncturemethod of blood, the blood will be collected in the device left at roomtemperature4h,3000rpm, centrifugal15min, the serum was collected, theserum collected in a water bath at56℃for30min to inactivate, and thenin the clean the station by0.22pm microporous membrane filtersterilization, the serum to a clean tube, set at-20℃, formulated into aculture medium containing serum concentration required when needed.2.CCK-8method to check the inhibitory drugs on serumconcentrations of five HepC2cell proliferation, to find the best andeffective serum concentration of the drug the NHS.3.NHS HepG2cells after administration of the drug-containingserum48h, AO/EB and DAPI staining and morphological changesobserved under a fluorescence microscope.4.Impact of flow cytometry AnnexinV-FITC/PI double staining NHScontaining serum on HepG2cell cycle and apoptosis percentage.5.Western blotting detection NHS containing serum on HepG2cellsP53Bcl-2effect on gene expression in.6.Immunohistochemistry was used to detect the impact of the threeNHS containing serum drug concentrations effective in HepG2cellsBcl-2, Bax and proliferating cell nuclear antigen (PCNA) expression.7.Pre-treatment serum-containing drugs, the relative expressionBcl-2and Bax mRNA in HepG2cells after Real time RT-PCR testing. Results：1.CTX group and NHS various concentrations of taurine ginsengsignificantly inhibited cell proliferation,and with increasing concentrationand action time, the number and speed of proliferation of HepG2cellsdecreased,NHS articipate each concentration group with the controlgroup, the difference was statistically significance (P<0.05,P<0.01).2.By fluorescence microscopy to observe the typical morphological changes:serum containing part of the nucleus presents crease like, corrugated,nuclear chromatin with orange,some chromatin appearscondensed state;CTX serum containing group,20%NHS containing serum group visible.a large number of different levels of necrosis and apoptosis, the cell size smaller, nuclear membrane disappears,theincrease in cytoplasmic vacuoles, organelles fewer nuclear chromatin condensation,irregular,visible nuclear fragmentation, resulting in apoptosis body, and training longer, more significant change.3. HepG2cells were analyzed by flow cytometry showed significantsubdiploid peak, and the number of apoptotic cells increases the doseincreases. HepG2cells containing serum CTX,5%NHS containing serum,10%NHS containing serum and20%NHS containing serum48h afteradministration, the proportion of apoptotic cells were30.13%,11.05%,19.76%and28.32%.Cell cycle analysis shows that NHS containingserum group so that prevents cell growth in the G0/G1phase.4.Western blotting showed that: Compared with the control group,the NHS reference serum containing acting drugs in HepG2cells reality,the expression of Bcl-2protein was significantly decreased (P<0.05), asignificant increase in the expression of P53protein (P<0.05), and thereis a significant difference (P<0.05) between the two groups compared.5.Immunohistochemical results showed that: with the increase in serum concentrations of NHS administration including, PCNALIgradually decreased, indicating that NHS containing serum inhibited theproliferation of HepG2tumor cells, and with the NHS with increasedserum concentrations of the drug can significantly reduce HepG2cells thepositive expression rate of the Bcl-2protein,Bax protein was expressed ina strong,20%NHS, CTX group and10%NHS positive expression rateswere56.89%,58.33%,44.73%, compared with the control group, with asignificant difference (P<0.01, P<0.05).6. Real-time PCR to detect the expression of CT values, the analysisof each factor related apoptosis relative expression levels of mRNAdisplay, along with the expression level of NHS drug-containing serumconcentration increased Bax mRNA increased, whereas the expression ofBcl-2mRNA levels but reduced.Conclusion：1. The NHS containing serum can inhibit the proliferation of HepG2cells in a certain extent.2. The NHS containing serum can induce apoptosis in HepG2cells.3. The NHS drug-containing serum induced HepG2apoptosis of oneof the mechanisms may be upregulated the expression of pro-apoptoticgenes Bax and P53, downregulate the expression of Bcl-2related.更多还原