复方中药合剂抗HCMV感染阳性胶质瘤干细胞作用及机制的研究

发布时间：2018-05-25 04:33

本文选题：人巨细胞病毒 + 胶质瘤干细胞　；参考：《青岛大学》2017年硕士论文

【摘要】：目的1.观察该复方中草药合剂在体外对HCMV感染的U251胶质瘤干细胞(GSCS)增殖及凋亡的影响。2.探讨该复方中草药合剂及联合更昔洛韦在体内对HCMV感染的U251GSCS移植瘤的生长及其血管生成的影响。方法1.根据莪术、冬凌草、白花蛇舌草抗病毒抗肿瘤特性制备抗胶质瘤复方中草药配方,将三种中草药提取物溶于培养基中,制备不同浓度的复方中草药合剂。2.用HCMV AD169毒株感染人胚肺成纤维细胞(HELF)进行病毒扩增,并用空斑定量法测HCMV滴度。3.用单克隆培养法从U251胶质瘤细胞系中分离出GSCs,用流式细胞仪检测U251GSCS中CD133+细胞的比例。4.U251GSCS接种HCMV(MOI=1),实验组加入不同药物浓度复方中草药合剂,对照组加入相同体积的培养基。培养48h后,用MTT法检测各组细胞活性。5.用RT-PCR检测各组细胞中HCMV基因IE-1、抑制凋亡基因Bcl-2及促进凋亡基因Bax m RNA的表达,Western blot检测各组Bcl-2及Bax蛋白表达的情况。6.将感染HCMV的U251GSCS移植到裸鼠颅内14d后,将裸鼠随机分为复方中草药合剂组、更昔洛韦组、复方中草药合剂联合更昔洛韦组及空白对照组。采用灌胃给药的方式治疗裸鼠,14天后处死裸鼠,取出脑组织中的肿瘤组织做病理检查。7.用免疫组化检测肿瘤中IE-1、CD34蛋白表达,并计算肿瘤微血管密度(MVD),RT-PCR检测肿瘤中Bcl-2、Bax m RNA的表达。结果1.U251GSCS在无血清培养基中悬浮生长,细胞呈球形,流式细胞仪结果显示,U251GSCS中CD133+细胞数达95%以上。2.MTT结果显示,复方中草药合剂可抑制细胞增殖,随着复方中草药合剂浓度及作用时间的增加,HCMV感染的U251GSCS活性降低(P0.05)。3.软琼脂克隆形成实验结果表明,该复方中草药合剂可明显抑制HCMV感染的U251GSCS的克隆形成,且随着药物浓度的增加,抑制效果越明显(P0.01)。4.RT-PCR结果显示,HCMV感染的U251GSCS与不同浓度复方中草药合剂培养48h后,细胞内促凋亡基因Bax m RNA的表达量增加,HCMV基因IE-1和抑凋亡基因Bcl-2m RNA的表达量减少(P0.05)。5.Western blot结果显示,HCMV感染的U251GSCS在不同浓度复方中草药合剂作用48h后,Bax蛋白表达量随着药物浓度的增加而增多,而Bcl-2蛋白表达量随着药物浓度的增加而减少(P0.05)。6.复方中草药合剂和更昔洛韦均能减少HCMV感染的U251GSCS移植瘤微血管的生成(P0.05),联合用药抑制移植瘤微血管生成较单独用药组效果明显,二者无交互作用(P0.05)。7.RT-PCR结果显示,复方中草药合剂和更昔洛韦能抑制移植瘤细胞内Bcl-2 m RNA的表达(P0.05),并促进Bax m RNA的表达(P0.05)。联合用药组Bcl-2 m RNA相对表达量明显低于复方中药组、更昔洛韦组和对照组(P0.05),联合用药组Bax mRNA相对表达量明显高于复方中药组、更昔洛韦组和对照组(P0.05)。结论1.该复方中草药合剂在体外能抑制HCMV感染的U251GSCS的增殖并诱导其凋亡。2.该复方中草药合剂可通过抑制肿瘤血管生成、诱导胶质瘤细胞凋亡从而抑制HCMV感染的U251GSCS原位移植瘤的生长。
[Abstract]:Objective 1. To observe the effect of compound Chinese herbal medicine mixture on proliferation and apoptosis of U251 glioma stem cells infected with HCMV in vitro. To investigate the effect of the compound Chinese herbal medicine mixture and ganciclovir on the growth and angiogenesis of U251GSCS transplanted tumor infected with HCMV in vivo. Method 1. According to the antiviral and antitumor properties of Rhizoma Curcumae, oridox and Euphorbia albicans, the compound Chinese herbal medicine formula of anti-glioma compound was prepared. The extracts of three kinds of Chinese herbal medicines were dissolved in the medium, and the compound Chinese herbal medicine mixture of different concentrations was prepared. Human embryonic lung fibroblast (HELF) was infected with HCMV AD169 strain to amplify the virus, and the titer of HCMV was measured by plaque quantitative method. GSCs were isolated from U251 glioma cell lines by monoclonal culture. The proportion of CD133 cells in U251GSCS was detected by flow cytometry. After culture for 48 h, the cell activity of each group was detected by MTT assay. The expression of HCMV gene IE-1, inhibiting apoptosis gene Bcl-2 and promoting apoptosis gene Bax m RNA were detected by RT-PCR. The expression of Bcl-2 and Bax protein was detected by Western blot. After transplanting U251GSCS infected with HCMV into the brain of nude mice for 14 days, nude mice were randomly divided into three groups: compound Chinese herbal medicine mixture group, ganciclovir group, compound Chinese herbal medicine mixture combined with ganciclovir group and blank control group. The nude mice were killed 14 days later by intragastric administration of drugs, and the tumor tissues in brain tissue were taken out for pathological examination. Immunohistochemical staining was used to detect the expression of IE-1pCD34 protein, and the expression of Bcl-2mBax m RNA was detected by RT-PCR in tumor microvessel density (MVD). Results 1.U251GSCS was suspended in serum-free medium and the cells were globular. The results of flow cytometry showed that the number of CD133 cells in U251 GSCS was over 95%. 2. The results showed that compound Chinese herbal medicine mixture could inhibit cell proliferation. With the increase of the concentration and action time of compound Chinese herbal medicine mixture, the U251GSCS activity of HCMV infection decreased P0.05. 3. The results of soft Agar clone formation test showed that the compound Chinese herbal medicine mixture could significantly inhibit the U251GSCS clone formation of HCMV infection, and with the increase of drug concentration, The more obvious the inhibitory effect was, the more obvious was the result of RT-PCR. The results of RT-PCR showed that U251GSCS infected with different concentrations of Chinese herbal medicine were cultured for 48 hours. The expression of Bax m RNA and Bcl-2m RNA decreased P0.05. 5. Western blot results showed that the expression of U251GSCS with different concentrations of Chinese herbal medicine mixture at different concentrations was increased with the increase of the expression of IE-1 and Bcl-2m RNA. 5. The results showed that the expression of Bax-Bax protein increased with the treatment of different concentrations of Chinese herbal medicine mixture for 48 h. With the increase of drug concentration, However, the expression of Bcl-2 protein decreased with the increase of drug concentration. Compound Chinese herbal medicine mixture and ganciclovir could reduce the formation of microvessel in U251GSCS transplanted tumor infected with HCMV. The effect of combined treatment on inhibiting microangiogenesis of transplanted tumor was more obvious than that of single drug group. The results of RT-PCR showed that there was no interaction between the two groups. Compound Chinese herbal medicine mixture and ganciclovir could inhibit the expression of Bcl-2 m RNA in transplanted tumor cells and promote the expression of Bax m RNA. The relative expression of Bcl-2 m RNA in the combined treatment group was significantly lower than that in the compound Chinese medicine group, and the relative expression of Bax mRNA in the ganciclovir group and the control group was significantly higher than that in the control group. Conclusion 1. The compound Chinese herbal medicine mixture can inhibit the proliferation and induce apoptosis of U251GSCS infected with HCMV in vitro. The compound Chinese herbal medicine mixture can inhibit the growth of HCMV infected U251GSCS orthotopic transplantation tumor by inhibiting tumor angiogenesis and inducing glioma cell apoptosis.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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