本文關(guān)鍵詞： 膠質(zhì)瘤 免疫治療 疫苗　出處：《西南醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:本研究探討以同種異基因型的C6膠質(zhì)瘤細(xì)胞及其裂解物與同基因型的9L膠質(zhì)瘤細(xì)胞裂解物作為免疫原的疫苗對(duì)大鼠膠質(zhì)瘤的治療作用,以及經(jīng)該疫苗治療后大鼠膠質(zhì)瘤組織的侵襲力變化情況,以期為膠質(zhì)瘤的免疫治療提供更多的可行性方案。方法:(1)采用立體定向技術(shù),將1×10~6/10μl個(gè)9L膠質(zhì)瘤細(xì)胞懸液注入Fisher 344大鼠右側(cè)尾狀核區(qū),建立9L/Fisher 344大鼠膠質(zhì)瘤原位模型。(2)接種后的大鼠隨機(jī)分為三組:空白組,二十萬(wàn)單位治療組,三十萬(wàn)單位治療組,每組各10只。其中空白組大鼠不接受治療;二十萬(wàn)單位治療組大鼠皮下注射以C6細(xì)胞(2×105個(gè))、C6細(xì)胞裂解物(含2×105個(gè)細(xì)胞)及9L細(xì)胞裂解物(含2×105個(gè)細(xì)胞)作為免疫原的疫苗;三十萬(wàn)單位治療組大鼠皮下注射以C6細(xì)胞(3×105個(gè))、C6細(xì)胞裂解物(含3×105個(gè)細(xì)胞)及9L細(xì)胞裂解物(含3×105個(gè)細(xì)胞)作為免疫原的疫苗。所有治療均于接種9L細(xì)胞后第1天進(jìn)行。(3)以50天為大鼠生存觀察期,記錄其生存狀況及生存時(shí)間。于接種9L細(xì)胞后第10、20、30、40、50天對(duì)所有大鼠行MRI檢查,動(dòng)態(tài)觀察大鼠膠質(zhì)瘤體積變化情況。接種9L細(xì)胞后第20天,各組隨機(jī)處死1只大鼠行HE染色及免疫組化Podoplanin、Fascin染色,以觀察大鼠膠質(zhì)瘤組織病理及侵襲能力改變情況。結(jié)果:(1)以50天為大鼠生存觀察期,空白組的生存率為0;二十萬(wàn)單位治療組大鼠的生存率為89%;三十萬(wàn)單位治療組大鼠的生存率為100%。治療組大鼠生存時(shí)間比空白組顯著延長(zhǎng),差異有統(tǒng)計(jì)學(xué)意義(P0.05);二十萬(wàn)單位治療組與三十萬(wàn)單位治療組之間比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。(2)所有大鼠增強(qiáng)MRI檢查均可見(jiàn)右側(cè)尾狀核區(qū)明顯強(qiáng)化,本次實(shí)驗(yàn)制模成功率為100%。MRI動(dòng)態(tài)檢查發(fā)現(xiàn),隨瘤齡增加,空白組大鼠膠質(zhì)瘤呈現(xiàn)持續(xù)增大,而治療組大鼠膠質(zhì)瘤呈現(xiàn)先增大后減小的生長(zhǎng)趨勢(shì)。接種9L細(xì)胞第20天時(shí),計(jì)算各組腫瘤體積:空白組大鼠膠質(zhì)瘤體積為(451.25±9.36)mm3,二十萬(wàn)單位治療組大鼠膠質(zhì)瘤體積為(122.52±22.30)mm3,三十萬(wàn)單位治療組大鼠膠質(zhì)瘤體積為(112.06±17.97)mm3。單因素方差分析證明空白組大鼠膠質(zhì)瘤體積明顯大于治療組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);二十萬(wàn)單位治療組與三十萬(wàn)單位治療組之間比較,差異無(wú)統(tǒng)計(jì)意義(P0.05)。(3)HE染色可見(jiàn),空白組大鼠膠質(zhì)瘤細(xì)胞密度高,排列緊密,呈巢狀,部分區(qū)域伴有出血、壞死及新生血管;治療組大鼠腦膠質(zhì)瘤組織的腫瘤細(xì)胞密度稍低,典型巢狀排列及新生血管少見(jiàn)。免疫組化Podoplanin及Fascin染色可見(jiàn),空白組大鼠Podoplanin及Fascin呈強(qiáng)陽(yáng)性表達(dá),而在治療組中,Podoplanin及Fascin表達(dá)均降低,且Podoplanin染色的降低程度比Fascin更加顯著;二十萬(wàn)單位治療組與三十萬(wàn)單位治療組之間相比較,均能觀察到少量的膠質(zhì)瘤細(xì)胞表達(dá)Podoplanin及Fascin,兩者之間無(wú)明顯差異。結(jié)論:本研究成功建立9L/Fisher 344大鼠腦膠質(zhì)瘤模型,并于接種后第1天予以同種異基因型的C6膠質(zhì)瘤細(xì)胞及其裂解物與同基因型的9L膠質(zhì)瘤細(xì)胞裂解物作為免疫原的疫苗進(jìn)行治療。實(shí)驗(yàn)結(jié)果證明了該疫苗能夠打破荷瘤機(jī)體對(duì)自身腫瘤的免疫耐受,誘導(dǎo)荷瘤機(jī)體的抗腫瘤免疫反應(yīng),防止免疫逃避的發(fā)生,延長(zhǎng)荷瘤大鼠生存時(shí)間并降低膠質(zhì)瘤組織侵襲力。
[Abstract]:Objective: To study the 9L glioma cell lysis with allogeneic type C6 glioma cells and lysates with the same genotype as vaccine immunogens on rat glioma treatment, as well as by the vaccine after treatment of rat glioma tissue invasion force change situation, in order to provide feasibility of more for immunotherapy of glioma. Methods: (1) by stereotactic technique, 1 x 10~6/10 L 9L glioma cell suspension was injected into the right caudate nucleus of 344 Fisher rats, 344 rats to establish 9L/Fisher glioma orthotopic model. (2) the rats were randomly divided into after the three groups: blank control group, treatment group two hundred thousand units, three hundred thousand units in the treatment group, 10 rats in each group. The rats in the blank group without treatment; two hundred thousand treated rats by subcutaneous injection of C6 cells (2 * 105), C6 cell lysates (containing 2 x 105 cells) and 9L cell Lysates (containing 2 x 105 cells) as vaccine immunogens; three hundred thousand treated rats by subcutaneous injection of C6 cells (3 * 105), C6 cell lysates (containing 3 x 105 cells) and 9L cell lysates (containing 3 x 105 cells) as vaccine immunogens all treatment was performed in first days after inoculation of 9L cells. (3) for a 50 day rat survival was observed, recorded the survival time in 10,20,30,40,50 days after inoculation of 9L cells of all rats were examined with MRI, the dynamic observation of rat glioma volume changes. Twentieth days were inoculated with 9L cells, 1 rats in each group were sacrificed for HE staining and immunohistochemical staining to observe Podoplanin, Fascin, glioma invasion and pathological changes in rats. Results: (1) to 50 days for the survival of rats after the observation period, the survival rate of control group was 0; two hundred thousand in the treatment group mice survival rate was 89%; thirty Survival million units of rats in the treatment group was 100%. treatment group rats survival time was significantly longer than that in control group, the difference was statistically significant (P0.05); two hundred thousand units and three hundred thousand units in treatment group between the treatment groups, the difference was not statistically significant (P0.05). (2) all rats enhanced MRI examination showed the right caudate nuclear area enhancement, the experiment system of the success rate of model was found for the 100%.MRI dynamic inspection, with the increase of tumor age, blank group rat glioma has continued to increase, while the treatment group rat glioma growth showed a trend of first increasing and then decreasing. 9L cells were inoculated twentieth days, tumor volume were calculated: blank group the glioma volume (451.25 + 9.36) mm3, two hundred thousand units of volume treatment of glioma rats for (122.52 + 22.30) mm3, three hundred thousand units of volume treatment of glioma rats for (112.06 + 17.97) mm3. single factor variance analysis show that the blank group The rat glioma volume was significantly greater than the treatment group, the difference was statistically significant (P0.05); two hundred thousand units and three hundred thousand units in treatment group between the treatment groups, the difference was not statistically significant (P0.05). (3) HE staining: blank group rat glioma cells with high density, arranged closely, nests, part area with hemorrhage, necrosis and tumor angiogenesis; treatment group rat brain glioma tumor cell density was low, the typical nests and neovascularization rare. Immunohistochemical staining of Podoplanin and Fascin, the rats in the blank group Podoplanin and Fascin showed strong positive expression in the treatment group, the expression of Podoplanin and Fascin decreased, and Podoplanin staining decreased more significantly than Fascin; two hundred thousand unit and three hundred thousand unit treatment group between the treatment groups were compared and observed in a small number of glioma cells expressed Podoplanin and Fascin, both No significant difference. Conclusion: This study successfully established 9L/Fisher rat brain glioma model 344, 9L glioma cell lysis and allogeneic type in first days after inoculation of C6 glioma cells and lysates with the same genotype as vaccine immunogens for treatment. The experimental results show that the the vaccine can break tumor immune tolerance to tumor, induce antitumor immune response of tumor bearing mice, prevent immune escape, prolong the survival time of tumor bearing rats and reduce glioma invasiveness.

【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41

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