【摘要】： 目的:卵巢癌是一種常見(jiàn)的,死亡率極高的婦科惡性腫瘤,目前主要采用手術(shù)、放療、化療相結(jié)合的治療方法,雖治療手段有了較大進(jìn)步,但療效并不樂(lè)觀。惡性腫瘤的發(fā)生發(fā)展與機(jī)體的免疫功能低下有關(guān),目前免疫基因治療越來(lái)越受到重視。CD40配體(CD40L)是一種免疫調(diào)節(jié)分子,CD40L與CD40結(jié)合能有效刺激T細(xì)胞增殖分化,增強(qiáng)其特異性殺傷腫瘤細(xì)胞的作用,還可誘導(dǎo)T細(xì)胞分泌細(xì)胞因子,從而發(fā)揮抗腫瘤作用。本課題旨在建立小鼠肝脾轉(zhuǎn)移動(dòng)物模型的基礎(chǔ)上,探討轉(zhuǎn)染CD40L基因的卵巢癌細(xì)胞株OVHM(CD40L-OVHM)脾臟接種后體內(nèi)抗腫瘤效應(yīng)的機(jī)制,為CD40L的臨床應(yīng)用提供實(shí)驗(yàn)依據(jù)。 方法: 1用MTT法測(cè)定CD40L-OVHM細(xì)胞、OVHM細(xì)胞和空載體DNA-pMKITneo-OVHM細(xì)胞的生長(zhǎng)曲線,應(yīng)用倒置顯微鏡觀察三種細(xì)胞的形態(tài)。 2用流式檢測(cè)儀檢測(cè)CD40L在CD40L-OVHM細(xì)胞和OVHM細(xì)胞的表達(dá)情況。 3用HE染色法驗(yàn)證經(jīng)小鼠脾內(nèi)接種OVHM細(xì)胞建立肝轉(zhuǎn)移模型的可行性。 4經(jīng)小鼠脾臟內(nèi)接種腫瘤細(xì)胞建立肝轉(zhuǎn)移模型分為四組:OVHM細(xì)胞組、空載體DNA-pMKITneo-OVHM細(xì)胞組、CD40L-OVHM細(xì)胞組及注射PBS液組。于小鼠脾臟接種腫瘤細(xì)胞后第10天用流式細(xì)胞儀分析不同組脾細(xì)胞CD11c分子的表達(dá)情況來(lái)反映樹(shù)突狀細(xì)胞(DC)的數(shù)量。于小鼠脾臟接種腫瘤細(xì)胞后第10天用MTT法檢測(cè)不同組脾臟殺傷性T細(xì)胞(CTL)的殺傷活性。于小鼠脾臟接種腫瘤細(xì)胞后第14天用ELISA法檢測(cè)不同組小鼠外周血血清中細(xì)胞因子(IFN-γ、TNF-α、IL-4、IL-10和IL-12)的含量。于小鼠脾臟接種腫瘤細(xì)胞后第14天稱量小鼠肝臟和脾臟的重量及觀察肝臟轉(zhuǎn)移瘤結(jié)節(jié)的情況。最后每組余下10只小鼠用于觀察生存期。 結(jié)果: 1鏡下觀察OVHM細(xì)胞、CD40L-OVHM細(xì)胞和DNA-pMKITneo-OVHM細(xì)胞三種細(xì)胞形態(tài)無(wú)明顯區(qū)別,測(cè)定三種細(xì)胞的生長(zhǎng)增殖情況無(wú)明顯差異。 2 CD40L-OVHM細(xì)胞表面大量表達(dá)CD40L分子,而OVHM細(xì)胞未見(jiàn)CD40L表達(dá)。 3小鼠脾臟內(nèi)接種腫瘤細(xì)胞OVHM細(xì)胞后第14天脾臟接種局部出現(xiàn)原發(fā)腫瘤,肝臟表面可見(jiàn)大量轉(zhuǎn)移瘤結(jié)節(jié),病理學(xué)證實(shí)小鼠肝臟和脾臟的腫瘤結(jié)節(jié)的病理學(xué)形態(tài)結(jié)構(gòu)相似,可見(jiàn)肝脾轉(zhuǎn)移動(dòng)物模型建立成功。 4接種OVHM細(xì)胞、DNA-pMKITneo-OVHM細(xì)胞和CD40L-OVHM細(xì)胞后10天的小鼠脾細(xì)胞中CD11c陽(yáng)性細(xì)胞率均明顯高于PBS對(duì)照組(P0.05),CD40L-OVHM細(xì)胞組小鼠的脾細(xì)胞中CD11c陽(yáng)性細(xì)胞率(20.30±1.23%)與DNA-pMKITneo-OVHM細(xì)胞組和OVHM細(xì)胞組小鼠脾細(xì)胞相比較(10.69±0.89%和10.56±0.85%)有顯著性提高(P0.05)。 5不同組小鼠的脾臟細(xì)胞毒性T細(xì)胞(CTL)殺傷活性檢測(cè)結(jié)果顯示:CD40L-OVHM細(xì)胞組、OVHM細(xì)胞組和DNA-pMKITneo-OVHM細(xì)胞組的小鼠脾細(xì)胞CTL殺傷率均高于PBS組(P0.05),CD40L-OVHM組對(duì)OVHM細(xì)胞的殺傷能力明顯高于OVHM組和DNA-pMKITneo-OVHM組(P0.05),OVHM組和DNA-pMKITneo-OVHM組間無(wú)顯著性差異(P0.05)。 6 CD40L-OVHM組血清TNF-α、IL-12和IFN-γ濃度明顯高于DNA-pMKITneo-OVHM細(xì)胞組和OVHM細(xì)胞組(P0.05);CD40L-OVHM組血清IL-4和IL-10濃度明顯低于OVHM組和DNA-pMKITneo-OVHM組(P0.05)。OVHM組和DNA-pMKITneo-OVHM組間血清TNF-α、IL-12、IFN-γ、IL-4、IL-10濃度均無(wú)明顯差異(P0.05)。 7小鼠脾內(nèi)接種腫瘤細(xì)胞發(fā)生肝轉(zhuǎn)移和脾臟原發(fā)瘤情況:小鼠脾臟接種腫瘤細(xì)胞后第14天,DNA-pMKITneo-OVHM細(xì)胞組和OVHM細(xì)胞組小鼠均出現(xiàn)肝轉(zhuǎn)移,脾臟均有原發(fā)瘤,肝臟平均重量分別為3.07±0.19g,3.10±0.21g;脾臟平均重量分別為1.25±0.14g,1.23±0.16g,兩組之間無(wú)顯著性差異。CD40L-OVHM細(xì)胞組小鼠中有3只小鼠發(fā)生了肝轉(zhuǎn)移(3/10),肝臟平均重量為1.45±0.14g;所有接種CD40L-OVHM細(xì)胞的小鼠脾臟均出現(xiàn)原發(fā)瘤,脾臟平均重量為0.58±0.10g。CD40L-OVHM細(xì)胞組小鼠的肝臟和脾臟重量均輕于OVHM細(xì)胞組和DNA-pMKITneo-OVHM細(xì)胞組肝脾的重量(P0.05),但與PBS對(duì)照組相比,OVHM細(xì)胞組、DNA-pMKITneo-OVHM細(xì)胞組和CD40L-OVHM細(xì)胞組的小鼠肝臟和脾臟重量均明顯增加(P0.05)。 8對(duì)照組PBS組小鼠在試驗(yàn)期間無(wú)一死亡,OVHM細(xì)胞組和DNA-pMKITneo-OVHM細(xì)胞組的小鼠在試驗(yàn)觀察期(65天)全部死亡,平均生存期分別為17±1天和18±1天;CD40L-OVHM細(xì)胞組的小鼠到實(shí)驗(yàn)結(jié)束時(shí)有6只死亡,其余4只在結(jié)束實(shí)驗(yàn)時(shí)仍然存活,平均生存期為58±3天,明顯長(zhǎng)于OVHM細(xì)胞組和DNA-pMKITneo-OVHM細(xì)胞組(P0.05)。 結(jié)論: 1通過(guò)建立小鼠肝脾轉(zhuǎn)移動(dòng)物模型證實(shí)表達(dá)CD40L的OVHM細(xì)胞(CD40L-OVHM)的體內(nèi)抑瘤作用明顯。 2表達(dá)CD40L的OVHM細(xì)胞(CD40L-OVHM)能夠有效地促進(jìn)脾臟DC的增殖和分化,增強(qiáng)脾臟細(xì)胞毒性T細(xì)胞(CTL)的特異性殺傷活性,增強(qiáng)機(jī)體局部抗腫瘤作用。 3表達(dá)CD40L的OVHM細(xì)胞(CD40L-OVHM)能夠有效地調(diào)節(jié)外周血細(xì)胞免疫功能,通過(guò)增強(qiáng)Th1型細(xì)胞的功能,抑制Th2型細(xì)胞的功能有效地調(diào)節(jié)了機(jī)體的Th1細(xì)胞和Th2細(xì)胞的平衡,增強(qiáng)了機(jī)體的抗腫瘤作用。 4為進(jìn)一步研究CD40L基因瘤苗及CD40L的臨床應(yīng)用提供了一定的試驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R737.31;R-332

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉秀均,戴W，

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