本文選題：人巨細胞病毒 + 膠質(zhì)瘤干細胞��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的1.觀察該復(fù)方中草藥合劑在體外對HCMV感染的U251膠質(zhì)瘤干細胞(GSCS)增殖及凋亡的影響。2.探討該復(fù)方中草藥合劑及聯(lián)合更昔洛韋在體內(nèi)對HCMV感染的U251GSCS移植瘤的生長及其血管生成的影響。方法1.根據(jù)莪術(shù)、冬凌草、白花蛇舌草抗病毒抗腫瘤特性制備抗膠質(zhì)瘤復(fù)方中草藥配方,將三種中草藥提取物溶于培養(yǎng)基中,制備不同濃度的復(fù)方中草藥合劑。2.用HCMV AD169毒株感染人胚肺成纖維細胞(HELF)進行病毒擴增,并用空斑定量法測HCMV滴度。3.用單克隆培養(yǎng)法從U251膠質(zhì)瘤細胞系中分離出GSCs,用流式細胞儀檢測U251GSCS中CD133+細胞的比例。4.U251GSCS接種HCMV(MOI=1),實驗組加入不同藥物濃度復(fù)方中草藥合劑,對照組加入相同體積的培養(yǎng)基。培養(yǎng)48h后,用MTT法檢測各組細胞活性。5.用RT-PCR檢測各組細胞中HCMV基因IE-1、抑制凋亡基因Bcl-2及促進凋亡基因Bax m RNA的表達,Western blot檢測各組Bcl-2及Bax蛋白表達的情況。6.將感染HCMV的U251GSCS移植到裸鼠顱內(nèi)14d后,將裸鼠隨機分為復(fù)方中草藥合劑組、更昔洛韋組、復(fù)方中草藥合劑聯(lián)合更昔洛韋組及空白對照組。采用灌胃給藥的方式治療裸鼠,14天后處死裸鼠,取出腦組織中的腫瘤組織做病理檢查。7.用免疫組化檢測腫瘤中IE-1、CD34蛋白表達,并計算腫瘤微血管密度(MVD),RT-PCR檢測腫瘤中Bcl-2、Bax m RNA的表達。結(jié)果1.U251GSCS在無血清培養(yǎng)基中懸浮生長,細胞呈球形,流式細胞儀結(jié)果顯示,U251GSCS中CD133+細胞數(shù)達95%以上。2.MTT結(jié)果顯示,復(fù)方中草藥合劑可抑制細胞增殖,隨著復(fù)方中草藥合劑濃度及作用時間的增加,HCMV感染的U251GSCS活性降低(P0.05)。3.軟瓊脂克隆形成實驗結(jié)果表明,該復(fù)方中草藥合劑可明顯抑制HCMV感染的U251GSCS的克隆形成,且隨著藥物濃度的增加,抑制效果越明顯(P0.01)。4.RT-PCR結(jié)果顯示,HCMV感染的U251GSCS與不同濃度復(fù)方中草藥合劑培養(yǎng)48h后,細胞內(nèi)促凋亡基因Bax m RNA的表達量增加,HCMV基因IE-1和抑凋亡基因Bcl-2m RNA的表達量減少(P0.05)。5.Western blot結(jié)果顯示,HCMV感染的U251GSCS在不同濃度復(fù)方中草藥合劑作用48h后,Bax蛋白表達量隨著藥物濃度的增加而增多,而Bcl-2蛋白表達量隨著藥物濃度的增加而減少(P0.05)。6.復(fù)方中草藥合劑和更昔洛韋均能減少HCMV感染的U251GSCS移植瘤微血管的生成(P0.05),聯(lián)合用藥抑制移植瘤微血管生成較單獨用藥組效果明顯,二者無交互作用(P0.05)。7.RT-PCR結(jié)果顯示,復(fù)方中草藥合劑和更昔洛韋能抑制移植瘤細胞內(nèi)Bcl-2 m RNA的表達(P0.05),并促進Bax m RNA的表達(P0.05)。聯(lián)合用藥組Bcl-2 m RNA相對表達量明顯低于復(fù)方中藥組、更昔洛韋組和對照組(P0.05),聯(lián)合用藥組Bax mRNA相對表達量明顯高于復(fù)方中藥組、更昔洛韋組和對照組(P0.05)。結(jié)論1.該復(fù)方中草藥合劑在體外能抑制HCMV感染的U251GSCS的增殖并誘導(dǎo)其凋亡。2.該復(fù)方中草藥合劑可通過抑制腫瘤血管生成、誘導(dǎo)膠質(zhì)瘤細胞凋亡從而抑制HCMV感染的U251GSCS原位移植瘤的生長。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.41

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