卵巢癌肝脾转移动物模型的建立及转染CD40L基因抗卵巢癌肝转移作用的体内实验研究
发布时间：2018-09-04 20:36
【摘要】： 目的:卵巢癌是一种常见的,死亡率极高的妇科恶性肿瘤,目前主要采用手术、放疗、化疗相结合的治疗方法,虽治疗手段有了较大进步,但疗效并不乐观。恶性肿瘤的发生发展与机体的免疫功能低下有关,目前免疫基因治疗越来越受到重视。CD40配体(CD40L)是一种免疫调节分子,CD40L与CD40结合能有效刺激T细胞增殖分化,增强其特异性杀伤肿瘤细胞的作用,还可诱导T细胞分泌细胞因子,从而发挥抗肿瘤作用。本课题旨在建立小鼠肝脾转移动物模型的基础上,探讨转染CD40L基因的卵巢癌细胞株OVHM(CD40L-OVHM)脾脏接种后体内抗肿瘤效应的机制,为CD40L的临床应用提供实验依据。 方法: 1用MTT法测定CD40L-OVHM细胞、OVHM细胞和空载体DNA-pMKITneo-OVHM细胞的生长曲线,应用倒置显微镜观察三种细胞的形态。 2用流式检测仪检测CD40L在CD40L-OVHM细胞和OVHM细胞的表达情况。 3用HE染色法验证经小鼠脾内接种OVHM细胞建立肝转移模型的可行性。 4经小鼠脾脏内接种肿瘤细胞建立肝转移模型分为四组:OVHM细胞组、空载体DNA-pMKITneo-OVHM细胞组、CD40L-OVHM细胞组及注射PBS液组。于小鼠脾脏接种肿瘤细胞后第10天用流式细胞仪分析不同组脾细胞CD11c分子的表达情况来反映树突状细胞(DC)的数量。于小鼠脾脏接种肿瘤细胞后第10天用MTT法检测不同组脾脏杀伤性T细胞(CTL)的杀伤活性。于小鼠脾脏接种肿瘤细胞后第14天用ELISA法检测不同组小鼠外周血血清中细胞因子(IFN-γ、TNF-α、IL-4、IL-10和IL-12)的含量。于小鼠脾脏接种肿瘤细胞后第14天称量小鼠肝脏和脾脏的重量及观察肝脏转移瘤结节的情况。最后每组余下10只小鼠用于观察生存期。 结果: 1镜下观察OVHM细胞、CD40L-OVHM细胞和DNA-pMKITneo-OVHM细胞三种细胞形态无明显区别,测定三种细胞的生长增殖情况无明显差异。 2 CD40L-OVHM细胞表面大量表达CD40L分子,而OVHM细胞未见CD40L表达。 3小鼠脾脏内接种肿瘤细胞OVHM细胞后第14天脾脏接种局部出现原发肿瘤,肝脏表面可见大量转移瘤结节,病理学证实小鼠肝脏和脾脏的肿瘤结节的病理学形态结构相似,可见肝脾转移动物模型建立成功。 4接种OVHM细胞、DNA-pMKITneo-OVHM细胞和CD40L-OVHM细胞后10天的小鼠脾细胞中CD11c阳性细胞率均明显高于PBS对照组(P0.05),CD40L-OVHM细胞组小鼠的脾细胞中CD11c阳性细胞率(20.30±1.23%)与DNA-pMKITneo-OVHM细胞组和OVHM细胞组小鼠脾细胞相比较(10.69±0.89%和10.56±0.85%)有显著性提高(P0.05)。 5不同组小鼠的脾脏细胞毒性T细胞(CTL)杀伤活性检测结果显示:CD40L-OVHM细胞组、OVHM细胞组和DNA-pMKITneo-OVHM细胞组的小鼠脾细胞CTL杀伤率均高于PBS组(P0.05),CD40L-OVHM组对OVHM细胞的杀伤能力明显高于OVHM组和DNA-pMKITneo-OVHM组(P0.05),OVHM组和DNA-pMKITneo-OVHM组间无显著性差异(P0.05)。 6 CD40L-OVHM组血清TNF-α、IL-12和IFN-γ浓度明显高于DNA-pMKITneo-OVHM细胞组和OVHM细胞组(P0.05);CD40L-OVHM组血清IL-4和IL-10浓度明显低于OVHM组和DNA-pMKITneo-OVHM组(P0.05)。OVHM组和DNA-pMKITneo-OVHM组间血清TNF-α、IL-12、IFN-γ、IL-4、IL-10浓度均无明显差异(P0.05)。 7小鼠脾内接种肿瘤细胞发生肝转移和脾脏原发瘤情况:小鼠脾脏接种肿瘤细胞后第14天,DNA-pMKITneo-OVHM细胞组和OVHM细胞组小鼠均出现肝转移,脾脏均有原发瘤,肝脏平均重量分别为3.07±0.19g,3.10±0.21g;脾脏平均重量分别为1.25±0.14g,1.23±0.16g,两组之间无显著性差异。CD40L-OVHM细胞组小鼠中有3只小鼠发生了肝转移(3/10),肝脏平均重量为1.45±0.14g;所有接种CD40L-OVHM细胞的小鼠脾脏均出现原发瘤,脾脏平均重量为0.58±0.10g。CD40L-OVHM细胞组小鼠的肝脏和脾脏重量均轻于OVHM细胞组和DNA-pMKITneo-OVHM细胞组肝脾的重量(P0.05),但与PBS对照组相比,OVHM细胞组、DNA-pMKITneo-OVHM细胞组和CD40L-OVHM细胞组的小鼠肝脏和脾脏重量均明显增加(P0.05)。 8对照组PBS组小鼠在试验期间无一死亡,OVHM细胞组和DNA-pMKITneo-OVHM细胞组的小鼠在试验观察期(65天)全部死亡,平均生存期分别为17±1天和18±1天;CD40L-OVHM细胞组的小鼠到实验结束时有6只死亡,其余4只在结束实验时仍然存活,平均生存期为58±3天,明显长于OVHM细胞组和DNA-pMKITneo-OVHM细胞组(P0.05)。 结论: 1通过建立小鼠肝脾转移动物模型证实表达CD40L的OVHM细胞(CD40L-OVHM)的体内抑瘤作用明显。 2表达CD40L的OVHM细胞(CD40L-OVHM)能够有效地促进脾脏DC的增殖和分化,增强脾脏细胞毒性T细胞(CTL)的特异性杀伤活性,增强机体局部抗肿瘤作用。 3表达CD40L的OVHM细胞(CD40L-OVHM)能够有效地调节外周血细胞免疫功能,通过增强Th1型细胞的功能,抑制Th2型细胞的功能有效地调节了机体的Th1细胞和Th2细胞的平衡,增强了机体的抗肿瘤作用。 4为进一步研究CD40L基因瘤苗及CD40L的临床应用提供了一定的试验基础。
[Abstract]:Objective: Ovarian cancer is a common gynecologic malignant tumor with high mortality. At present, it is mainly treated by surgery, radiotherapy and chemotherapy. Although the treatment methods have made great progress, the curative effect is not optimistic. CD40 ligand (CD40L) is an immunomodulatory molecule. The combination of CD40L and CD40 can effectively stimulate the proliferation and differentiation of T cells, enhance their specific killing effect on tumor cells, and induce T cells to secrete cytokines, thus playing an anti-tumor role. The mechanism of anti-tumor effect in vivo of ovarian cancer cell line OVHM (CD40L-OVHM) after spleen inoculation provides experimental basis for clinical application of CD40L.
Method:
1 The growth curves of CD40L-OVHM cells, OVHM cells and empty carrier DNA-pMKITneo-OVHM cells were measured by MTT method. The morphology of the three cells was observed by inverted microscope.
2 the expression of CD40L in CD40L-OVHM cells and OVHM cells was detected by flow cytometry.
3 HE staining method was used to verify the feasibility of establishing a liver metastasis model by inoculating OVHM cells in mice spleen.
4. The liver metastasis model was established by inoculating tumor cells into the spleen of mice and divided into four groups: OVHM cell group, empty vector DNA-pMKITneo-OVHM cell group, CD40L-OVHM cell group and PBS injection group. The cytotoxicity of splenic killer T cells (CTL) was detected by MTT on the 10th day after inoculation of tumor cells in mice spleen. The levels of cytokines (IFN-gamma, TNF-alpha, IL-4, IL-10 and IL-12) in peripheral blood of mice in different groups were detected by ELISA on the 14th day after inoculation of tumor cells in mice spleen. The weight of liver and spleen and the metastatic nodules of liver were measured on the 14th day after transplantation. The remaining 10 mice in each group were used to observe the survival time.
Result:
The morphology of OVHM cells, CD40L-OVHM cells and DNA-pMKITneo-OVHM cells were not significantly different, and the growth and proliferation of the three cells were not significantly different.
2 CD40L molecules were expressed on the surface of CD40L-OVHM cells, but no CD40L expression was found in OVHM cells.
3. On the 14th day after inoculating OVHM cells into the spleen of mice, primary tumors appeared locally, and a large number of metastatic nodules were seen on the surface of the liver. Pathological examination showed that the pathological structure of the tumor nodules in the liver and spleen of mice was similar, and the animal model of liver and spleen metastasis was established successfully.
The percentage of CD11c positive cells in spleen cells of mice inoculated with OVHM cells, DNA-pMKITneo-OVHM cells and CD40L-OVHM cells 10 days after inoculation was significantly higher than that of mice inoculated with PBS (P 0.05). The percentage of CD11c positive cells in spleen cells of mice inoculated with CD40L-OVHM cells (20.30+1.23%) was significantly higher than that of mice inoculated with DNA-pMKITneo-OVHM cells and OVHM cells (10.69+0.89). % and 10.56 + 0.85%) increased significantly (P0.05).
The cytotoxic activity of splenic cytotoxic T lymphocytes (CTL) in mice of different groups showed that the CTL killing rate of spleen cells of CD40L-OVHM cell group, OVHM cell group and DNA-pMKITneo-OVHM cell group was higher than that of PBS group (P 0.05). The killing ability of CD40L-OVHM group to OVHM cells was significantly higher than that of OVHM group and DNA-pMKITneo-OVHM group (P 0.05), VHM group and DNA-pMKITneo-OVHM group (P 0.05). There was no significant difference between group -pMKITneo-OVHM (P0.05).
The concentrations of serum TNF-a, IL-12 and IFN-y in 6 CD40L-OVHM group were significantly higher than those in DNA-pMKITneo-OVHM cell group and OVHM cell group (P 0.05); the concentrations of IL-4 and IL-10 in CD40L-OVHM group were significantly lower than those in OVHM group and DNA-pMKITneo-OVHM group (P 0.05). There was no significant difference between OVHM group and DNA-pMKITneo-OVHM group (P 0.05).In the meantime, it is necessary to study the relationship between the two.
Hepatic metastasis and primary splenic tumor in mice inoculated with tumor cells: On the 14th day after inoculation, liver metastasis occurred in mice inoculated with tumor cells in DNA-pMKITneo-OVHM and OVHM cell groups. Primary tumors were found in spleen, with the average weight of liver being 3.07 (+ 0.19 g) and 3.10 (+ 0.21 g), and the average weight of spleen being 1.25 (+ 0.14 g) and 1.23 (+ 0.21 g) respectively. There were 3 mice in CD40L-OVHM cell group with liver metastasis (3/10) and the average weight of liver was 1.45.14 g. All mice inoculated with CD40L-OVHM cells had primary tumors in their spleens. The average weight of liver and spleen in CD40L-OVHM cell group was 0.58.10 G. The liver and spleen weight of OVHM group, DNA-pMKITneo-OVHM cell group and CD40L-OVHM cell group were significantly higher than that of PBS control group (P 0.05).
None of the PBS mice in the control group died during the experiment. All the mice in the OVHM cell group and DNA-pMKITneo-OVHM cell group died during the observation period (65 days), with an average survival time of 17 [1 day] and 18 [1 day], respectively. Six mice in the CD40L-OVHM cell group died at the end of the experiment, and the other four mice still survived at the end of the experiment, with an average survival time. For 58 + 3 days, it was significantly longer than OVHM cell group and DNA-pMKITneo-OVHM cell group (P0.05).
Conclusion:
1. Establishment of an animal model of liver and spleen metastasis in mice confirmed that CD40L-expressing OVHM cells (CD40L-OVHM) had obvious tumor inhibition in vivo.
OVHM cells expressing CD40L (CD40L-OVHM) can effectively promote the proliferation and differentiation of splenic DC, enhance the specific killing activity of splenic cytotoxic T cells (CTL) and enhance the local anti-tumor effect of the body.
OVHM cells expressing CD40L (CD40L-OVHM) can effectively regulate the immune function of peripheral blood cells. By enhancing the function of Th1 cells and inhibiting the function of Th2 cells, the balance of Th1 cells and Th2 cells can be effectively regulated, and the anti-tumor effect of the body can be enhanced.
4 provide a basis for further research on the clinical application of CD40L gene vaccine and CD40L.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R737.31;R-332
[Abstract]:Objective: Ovarian cancer is a common gynecologic malignant tumor with high mortality. At present, it is mainly treated by surgery, radiotherapy and chemotherapy. Although the treatment methods have made great progress, the curative effect is not optimistic. CD40 ligand (CD40L) is an immunomodulatory molecule. The combination of CD40L and CD40 can effectively stimulate the proliferation and differentiation of T cells, enhance their specific killing effect on tumor cells, and induce T cells to secrete cytokines, thus playing an anti-tumor role. The mechanism of anti-tumor effect in vivo of ovarian cancer cell line OVHM (CD40L-OVHM) after spleen inoculation provides experimental basis for clinical application of CD40L.
Method:
1 The growth curves of CD40L-OVHM cells, OVHM cells and empty carrier DNA-pMKITneo-OVHM cells were measured by MTT method. The morphology of the three cells was observed by inverted microscope.
2 the expression of CD40L in CD40L-OVHM cells and OVHM cells was detected by flow cytometry.
3 HE staining method was used to verify the feasibility of establishing a liver metastasis model by inoculating OVHM cells in mice spleen.
4. The liver metastasis model was established by inoculating tumor cells into the spleen of mice and divided into four groups: OVHM cell group, empty vector DNA-pMKITneo-OVHM cell group, CD40L-OVHM cell group and PBS injection group. The cytotoxicity of splenic killer T cells (CTL) was detected by MTT on the 10th day after inoculation of tumor cells in mice spleen. The levels of cytokines (IFN-gamma, TNF-alpha, IL-4, IL-10 and IL-12) in peripheral blood of mice in different groups were detected by ELISA on the 14th day after inoculation of tumor cells in mice spleen. The weight of liver and spleen and the metastatic nodules of liver were measured on the 14th day after transplantation. The remaining 10 mice in each group were used to observe the survival time.
Result:
The morphology of OVHM cells, CD40L-OVHM cells and DNA-pMKITneo-OVHM cells were not significantly different, and the growth and proliferation of the three cells were not significantly different.
2 CD40L molecules were expressed on the surface of CD40L-OVHM cells, but no CD40L expression was found in OVHM cells.
3. On the 14th day after inoculating OVHM cells into the spleen of mice, primary tumors appeared locally, and a large number of metastatic nodules were seen on the surface of the liver. Pathological examination showed that the pathological structure of the tumor nodules in the liver and spleen of mice was similar, and the animal model of liver and spleen metastasis was established successfully.
The percentage of CD11c positive cells in spleen cells of mice inoculated with OVHM cells, DNA-pMKITneo-OVHM cells and CD40L-OVHM cells 10 days after inoculation was significantly higher than that of mice inoculated with PBS (P 0.05). The percentage of CD11c positive cells in spleen cells of mice inoculated with CD40L-OVHM cells (20.30+1.23%) was significantly higher than that of mice inoculated with DNA-pMKITneo-OVHM cells and OVHM cells (10.69+0.89). % and 10.56 + 0.85%) increased significantly (P0.05).
The cytotoxic activity of splenic cytotoxic T lymphocytes (CTL) in mice of different groups showed that the CTL killing rate of spleen cells of CD40L-OVHM cell group, OVHM cell group and DNA-pMKITneo-OVHM cell group was higher than that of PBS group (P 0.05). The killing ability of CD40L-OVHM group to OVHM cells was significantly higher than that of OVHM group and DNA-pMKITneo-OVHM group (P 0.05), VHM group and DNA-pMKITneo-OVHM group (P 0.05). There was no significant difference between group -pMKITneo-OVHM (P0.05).
The concentrations of serum TNF-a, IL-12 and IFN-y in 6 CD40L-OVHM group were significantly higher than those in DNA-pMKITneo-OVHM cell group and OVHM cell group (P 0.05); the concentrations of IL-4 and IL-10 in CD40L-OVHM group were significantly lower than those in OVHM group and DNA-pMKITneo-OVHM group (P 0.05). There was no significant difference between OVHM group and DNA-pMKITneo-OVHM group (P 0.05).In the meantime, it is necessary to study the relationship between the two.
Hepatic metastasis and primary splenic tumor in mice inoculated with tumor cells: On the 14th day after inoculation, liver metastasis occurred in mice inoculated with tumor cells in DNA-pMKITneo-OVHM and OVHM cell groups. Primary tumors were found in spleen, with the average weight of liver being 3.07 (+ 0.19 g) and 3.10 (+ 0.21 g), and the average weight of spleen being 1.25 (+ 0.14 g) and 1.23 (+ 0.21 g) respectively. There were 3 mice in CD40L-OVHM cell group with liver metastasis (3/10) and the average weight of liver was 1.45.14 g. All mice inoculated with CD40L-OVHM cells had primary tumors in their spleens. The average weight of liver and spleen in CD40L-OVHM cell group was 0.58.10 G. The liver and spleen weight of OVHM group, DNA-pMKITneo-OVHM cell group and CD40L-OVHM cell group were significantly higher than that of PBS control group (P 0.05).
None of the PBS mice in the control group died during the experiment. All the mice in the OVHM cell group and DNA-pMKITneo-OVHM cell group died during the observation period (65 days), with an average survival time of 17 [1 day] and 18 [1 day], respectively. Six mice in the CD40L-OVHM cell group died at the end of the experiment, and the other four mice still survived at the end of the experiment, with an average survival time. For 58 + 3 days, it was significantly longer than OVHM cell group and DNA-pMKITneo-OVHM cell group (P0.05).
Conclusion:
1. Establishment of an animal model of liver and spleen metastasis in mice confirmed that CD40L-expressing OVHM cells (CD40L-OVHM) had obvious tumor inhibition in vivo.
OVHM cells expressing CD40L (CD40L-OVHM) can effectively promote the proliferation and differentiation of splenic DC, enhance the specific killing activity of splenic cytotoxic T cells (CTL) and enhance the local anti-tumor effect of the body.
OVHM cells expressing CD40L (CD40L-OVHM) can effectively regulate the immune function of peripheral blood cells. By enhancing the function of Th1 cells and inhibiting the function of Th2 cells, the balance of Th1 cells and Th2 cells can be effectively regulated, and the anti-tumor effect of the body can be enhanced.
4 provide a basis for further research on the clinical application of CD40L gene vaccine and CD40L.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R737.31;R-332
【参考文献】
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1 刘秀均,戴W，
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