本文選題：VP3 + 基因克隆�。� 參考：《中南大學》2014年博士論文

【摘要】：光動力治療(Photodynamic therapy, PDT)是采用一定波長的光照射蓄積于腫瘤細胞內(nèi)的光敏劑,發(fā)生光物理化學反應(yīng),產(chǎn)生單態(tài)氧和自由基等其他活性物質(zhì)、破壞腫瘤微血管、增強機體免疫力,促進細胞凋亡以殺傷腫瘤細胞的一種非手術(shù)治療方法。然而PDT是對氧的依賴性及治療深度的局限性限制其在腫瘤治療中的廣泛應(yīng)用。近年來,隨著基因治療日益成為治療腫瘤的最有前景的手段之一,PDT與基因治療有機結(jié)合形成的PDT/基因治療可能是一種治療腫瘤的嶄新策略。本文將分章討論自殺基因VP3對鼻咽癌細胞的殺傷作用及體外體內(nèi)實驗驗證PDT聯(lián)合自殺基因VP3治療鼻咽癌的療效。 第一章自殺基因VP3對鼻咽癌CNE-2細胞生長、增殖及凋亡的影響 目的體外實驗研究VP3基因其對鼻咽癌細胞CNE-2生長、增殖、凋亡的影響,以求證實VP3對鼻咽癌細胞的凋亡誘導效應(yīng)。 方法采用脂質(zhì)體轉(zhuǎn)染法將構(gòu)建的PcDNA3.1(-)VP3-Myc(PVP3)及對照空載體質(zhì)粒PcDNA3.1(-)轉(zhuǎn)染鼻咽癌CNE-2細胞。通過熒光顯微鏡觀察質(zhì)粒轉(zhuǎn)染效率,CCK-8法檢測細胞生存率,RT-PCR、Western blot法檢測VP3表達效果,通過Annexin V檢測VP3對細胞凋亡的影響。 結(jié)果 1.VP3mRNA在野生型及空載體質(zhì)粒轉(zhuǎn)染組細胞中均無明顯表達,在PVP3質(zhì)粒轉(zhuǎn)染組中有明顯表達,相對表達量約1.27±0.24。VP3蛋白(apoptin)在野生型及空載體質(zhì)粒轉(zhuǎn)染組細胞中均無明顯表達,在PVP3質(zhì)粒轉(zhuǎn)染組中有明顯表達,相對表達量約0.22±0.06。 2.空載體質(zhì)粒轉(zhuǎn)染組在24h、48h、72h細胞存活率分別為97.6±1.4%,94.2±1.7%、92.3±2.1%；PVP3質(zhì)粒轉(zhuǎn)染組在24h、48h、72h細胞存活率分別為79.7±3.6%,61.5±2.3%、52.4±3.8%。PVP3質(zhì)粒轉(zhuǎn)染與空載體質(zhì)粒轉(zhuǎn)染組比較有明顯統(tǒng)計學差異(P0.001)。 3.空載體質(zhì)粒轉(zhuǎn)染組在24h、48h、72h細胞凋亡率分別為4.5±1.2%、5.1±0.8%、5.7±1.4%；PVP3質(zhì)粒轉(zhuǎn)染組在24h、48h、72h細胞凋亡率分別為11.6±2.4%、17.8±1.4%、27.3±3.3%。PVP3質(zhì)粒轉(zhuǎn)染與空載體質(zhì)粒轉(zhuǎn)染組比較有明顯統(tǒng)計學差異(P0.001)。 結(jié)論成功建立了VP3基因穩(wěn)定表達的鼻咽癌CNE-2細胞株,體外實驗證實了VP3能明顯抑制鼻咽癌CNE-2細胞的生長增殖,并能促進CNE-2細胞的凋亡,為后續(xù)實驗提供了研究依據(jù) 第二章光動力治療聯(lián)合VP3基因治療對鼻咽癌CNE-2細胞的體外實驗研究 目的擬對VP3聯(lián)合PDT對鼻咽癌CNE-2細胞進行體外實驗研究,以證實VP3聯(lián)合PDT對鼻咽癌CNE-2細胞的殺傷效果。 方法穩(wěn)定表達VP3基因的鼻咽癌細胞CNE-2及對照組細胞分別接受不同能量密度的PDT治療,CCK-8法檢測細胞生存率,Annexin V法、Tunnel法檢測細胞凋亡。 結(jié)果 1.不同能量密度的PDT處理后,PVP3組細胞存活率明顯低于空載體質(zhì)粒轉(zhuǎn)染組細胞,實驗組細胞在1、3、6.25J/cm2能量密度下生存率分別為0.533±0.036、0.312±0.027、0.171±0.032；對照組細胞在1、3、6.25J/cm2能量密度下生存率分別為0.816±0.058、0.570±0.044、0.308±0.047,兩組比較有明顯統(tǒng)計學差異(P0.001)。 2.Annexin V檢測顯示PVP3組細胞凋亡率明顯高于空載體質(zhì)粒轉(zhuǎn)染組細胞,實驗組細胞在1、3、6.25J/cm2能量密度下凋亡率分別為0.363±0.025、0.672±0.043、0.741±0.039；對照組細胞在1、3、6.25J/cm2能量密度下凋亡率分別為0.266±0.027、0.568±0.055、0.671±0.046,兩組比較有明顯統(tǒng)計學差異(P0.001)；Tunnel法檢測顯示實驗組細胞中大部分細胞膨脹,細胞核碎裂,對照組細胞膜較完整,部分細胞呈空泡狀,細胞核濃縮,呈塊狀,少量細胞細胞核碎裂。 結(jié)論細胞實驗研究發(fā)現(xiàn)與單純PDT相比,PVP3+PDT聯(lián)合治療能顯著抑制鼻咽癌CNE-2細胞的生長增殖,并且能顯著增強鼻咽癌CNE-2細胞的凋亡。 第三章VP3聯(lián)合光動力治療對鼻咽癌CNE-2細胞裸鼠移植瘤作用的實驗研究 目的動物實驗中比較VP3聯(lián)合PDT與單純PDT或VP3基因治療對鼻咽癌的療效,以進一步證實VP3聯(lián)合PDT對鼻咽癌的高效殺傷效果。 方法構(gòu)建裸鼠人鼻咽癌移植瘤模型,PDT處理后,測量并記錄腫瘤的大小、重量;HE染色比較各組移植瘤病理學特點；免疫組化檢測移植瘤中apoptin的表達。 結(jié)果 1.空載體質(zhì)粒組、PVP3質(zhì)粒組、CNE2/Mock+PDT和CNE2/VP3+PDT處理后0、3、7、14天時分別比較四組腫瘤體積,經(jīng)ANOVA方差分析,四組間腫瘤體積差異有顯著性意義,經(jīng)兩樣本獨立t檢驗方法進行兩兩比較,結(jié)果顯示CNE2/VP3+PDT處理組腫瘤體積與其他各組相比明顯減小(P0.001)；且CNE2/VP3組腫瘤體積較CNE2/Mock組明顯減小(P0.001)。 2.四組移植瘤重量差異有顯著性意義,經(jīng)LSD方法進行兩兩比較,CNE2/VP3+PDT處理組移植瘤重量與各對照組相比明顯減小(P0.001); CNE2/VP3組移植瘤重量較CNE2/Mock組明顯減小(P0.001)。 3.HE染色檢查發(fā)現(xiàn)CNE2/Mock組移植瘤腫瘤細胞形態(tài)正常,瘤體內(nèi)見毛細血管豐富,未見明顯壞死組織；CNE2/VP3組移植瘤腫瘤細胞形態(tài)正常,可見少許壞死區(qū)域；CNE2/Mock+PDT組移植瘤見大量腫瘤細胞變性及核固縮,伴有部分片狀壞死區(qū)域；CNE2/VP3+PDT組移植瘤見大片狀壞死區(qū)域,僅少量癌細胞島殘留。 4.免疫組化檢查發(fā)現(xiàn)CNE2/Mock組及CNE2/Mock+PDT組未見明顯apoptin蛋白表達；CNE2/VP3組及CNE2/VP3+PDT組apoptin蛋白強表達,且其主要在細胞核中表達。 結(jié)論利用基因治療或者光動力治療均能抑制鼻咽癌裸鼠移植瘤生長,而光動力治療聯(lián)合基因治療較單純的光動力治療或基因治療對鼻咽癌裸鼠移植瘤有更好的殺傷效果。光動力治療聯(lián)合基因治療為鼻咽癌的臨床治療提供了實驗基礎(chǔ)。
[Abstract]:Photodynamic therapy (Photodynamic therapy, PDT) is a kind of non operative treatment of tumor cells by irradiation of light sensitizers accumulated in tumor cells by light irradiation of a certain wavelength, producing light physical and chemical reactions, producing other active substances such as mono oxygen and free radicals, destroying tumor microvessels, enhancing the body's immunity and promoting cell apoptosis. However, PDT is the limitation of the dependence on oxygen and the limitation of the depth of treatment. It has been widely used in cancer treatment. In recent years, as gene therapy has become one of the most promising ways to treat cancer, the PDT/ gene therapy formed by the organic combination of PDT and gene therapy may be a new strategy for the treatment of tumors. This article will be divided into this article. This chapter discusses the killing effect of suicide gene VP3 on nasopharyngeal carcinoma cells and the effect of PDT combined suicide gene VP3 on nasopharyngeal carcinoma in vivo and in vitro.
Chapter 1 the effect of suicide gene VP3 on growth, proliferation and apoptosis of nasopharyngeal carcinoma CNE-2 cells
Objective to study the effect of VP3 gene on the growth, proliferation and apoptosis of nasopharyngeal carcinoma cell CNE-2 in vitro, in order to verify the apoptosis inducing effect of real VP3 on nasopharyngeal carcinoma cells.
Methods the transfected PcDNA3.1 (-) (-) VP3-Myc (PVP3) and PcDNA3.1 (-) were transfected into the CNE-2 cells of nasopharyngeal carcinoma by liposome transfection. The transfection efficiency of the plasmid was observed by the fluorescence microscope, the cell survival rate was detected by CCK-8 method. The expression of VP3 was detected by RT-PCR, Western blot, and the effect of Annexin V on the apoptosis was detected by Annexin V.
Result
There was no obvious expression of 1.VP3mRNA in the transfected group cells of wild type and unloaded body, and there was obvious expression in the transfection group of PVP3 plasmid. The relative expression of about 1.27 + 0.24.VP3 protein (apoptin) had no obvious expression in the cells of the wild type and unloaded body transfected group, and the expression of relative expression was about 0.22 + 0 in the PVP3 plasmid transfection group. Six
The survival rate of 24h, 48h and 72h cells was 97.6 + 1.4%, 94.2 + 1.7%, 92.3 + 2.1%, respectively. The survival rate of PVP3 plasmid transfected group in 24h, 48h, 72h cells was 79.7 + 3.6%, 61.5 + and 61.5 +, respectively, and 52.4 + 3.8%.PVP3 plasmid transfection was significantly different (P0.001) compared with the empty body transfection group (P0.001).
The apoptosis rate of 24h, 48h and 72h cells was 4.5 + 1.2%, 5.1 + 0.8%, 5.7 + 1.4%, respectively. The apoptosis rate of PVP3 plasmid transfected group in 24h, 48h, 72h cells was 11.6 + 2.4%, 17.8 + and 17.8 +, respectively, and 27.3 + 3.3%.PVP3 plasmid transfection was significantly different (P0.001) compared with the empty body transfection group (P0.001).
Conclusion the CNE-2 cell line of nasopharyngeal carcinoma with stable expression of VP3 gene was successfully established. In vitro experiments confirmed that VP3 could obviously inhibit the growth and proliferation of CNE-2 cells in nasopharyngeal carcinoma and promote the apoptosis of CNE-2 cells, which provides a basis for further research.
Second Zhang Guang dynamic therapy combined with VP3 gene therapy for nasopharyngeal carcinoma CNE-2 cells in vitro
Objective to study the effect of VP3 combined with PDT on nasopharyngeal carcinoma CNE-2 cells in vitro, in order to confirm the killing effect of VP3 combined with PDT on nasopharyngeal carcinoma CNE-2 cells.
Methods the CNE-2 of nasopharyngeal carcinoma cells expressing VP3 gene and the control group cells were treated with PDT with different energy density respectively. The cell survival rate was detected by CCK-8 method, Annexin V method and Tunnel method were used to detect the apoptosis.
Result
1. after PDT treatment with different energy density, the survival rate of cells in PVP3 group was significantly lower than that of the empty body mass transfected group. The survival rate of the cells in the experimental group was 0.533 + 0.036,0.312 + 0.027,0.171 0.032 respectively under the 1,3,6.25J/cm2 energy density, and the survival rate of the cells in the control group was 0.816 + 0.058,0.570 + 0.044,0.3 under the 1,3,6.25J/cm2 energy density, respectively. 08 + 0.047, there was a significant difference between the two groups (P0.001).
2.Annexin V detection showed that the apoptosis rate of the PVP3 group was significantly higher than that of the empty body mass transfected group. The apoptosis rate of the experimental group was 0.363 + 0.025,0.672 + 0.039 respectively under the 1,3,6.25J/cm2 energy density, and the apoptosis rate of the control group was 0.266 + 0.027,0.568 + 0.055,0.671 + 0.046, two, under the 1,3,6.25J/cm2 energy density, two There was significant difference between the groups (P0.001), and the Tunnel test showed that most of the cells in the experimental group were expanded, the nuclei were broken, the cell membrane of the control group was more complete, some cells were vacuolated, the nuclei were concentrated, and the cell nuclei were fragmented.
Conclusion compared with simple PDT, PVP3+PDT combined therapy can significantly inhibit the growth and proliferation of nasopharyngeal carcinoma CNE-2 cells, and can significantly enhance the apoptosis of nasopharyngeal carcinoma CNE-2 cells.
Third chapter VP3 combined photodynamic therapy on nasopharyngeal carcinoma CNE-2 cell xenograft in nude mice
Objective to compare the therapeutic effect of VP3 combined with PDT and simple PDT or VP3 gene therapy on nasopharyngeal carcinoma in order to further confirm the effect of VP3 combined with PDT on nasopharyngeal carcinoma.
Methods the model of human nasopharyngeal carcinoma transplanted in nude mice was constructed. After PDT treatment, the size and weight of the tumor were measured and recorded. The pathological features of the tumor were compared with HE staining, and the expression of apoptin in the transplanted tumor was detected by immunohistochemistry.
Result
1. empty body mass group, PVP3 plasmid group, CNE2/Mock+PDT and CNE2/VP3+PDT after 0,3,7,14 days were compared with four groups of tumor volume respectively. After ANOVA variance analysis, the difference of tumor volume between the four groups was significant, and the two samples were compared with the independent t test method for 22 comparison. The results showed that the volume of the tumor in the CNE2/VP3+PDT treatment group and the other groups were shown in the other groups. The ratio was significantly reduced (P0.001), and the volume of tumor in group CNE2/VP3 was significantly lower than that in group CNE2/Mock (P0.001).
2. the weight difference between the four groups was significant. Compared with the 22 comparison by the LSD method, the weight of the transplanted tumor in the CNE2/VP3+PDT treatment group was significantly lower than that of the control group (P0.001), and the weight of the transplanted tumor in the group CNE2/VP3 was significantly lower than that in the CNE2/Mock group (P0.001).
3.HE staining showed that the tumor cells of group CNE2/Mock were normal, and the blood capillary was rich in the tumor and no obvious necrotic tissue was found in the tumor. The tumor cells in group CNE2/VP3 were normal, and a little necrotic area was found. In group CNE2/Mock+PDT, a large number of tumor cell degeneration and nuclear fixation, accompanied by partial necrosis area, were found in group CNE; CNE In group 2/VP3+PDT, there were large necrotic areas and only a small number of cancer cell islands remained.
4. immunohistochemical examination showed that no obvious expression of apoptin protein was found in group CNE2/Mock and CNE2/Mock+PDT group, and the expression of apoptin protein in group CNE2/VP3 and CNE2/VP3+PDT group was strongly expressed, and it was mainly expressed in the nucleus.
Conclusion both gene therapy and photodynamic therapy can inhibit the growth of nasopharyngeal carcinoma transplanted tumor in nude mice. Photodynamic therapy combined with gene therapy has better killing effect on nude mice transplanted tumor of nasopharyngeal carcinoma than simple photodynamic therapy or gene therapy. The combination of photodynamic therapy and gene therapy provides the experimental basis for the clinical treatment of nasopharyngeal carcinoma.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R739.63

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相關(guān)期刊論文 前1條

1 馬麗萍,張文庚,曹國棟,王申五,張志義;轉(zhuǎn)促凋亡基因胃癌細胞的光動力學治療實驗研究[J];北京醫(yī)科大學學報;2000年03期

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