【作者】 王凯峰；

【导师】 叶胜龙； 吕丽娜； 钱诗光；

【作者基本信息】 复旦大学 ， 肿瘤学， 2005， 博士

【摘要】 研究目的 建立T淋巴细胞免疫功能重建模型,提供进行免疫与肝癌转移关系研究的实验平台。应用该模型研究T细胞免疫功能重建对小鼠高、低淋巴结转移潜能的肝癌的影响,同时研究影响肝癌转移的免疫学因素,为深入寻找肝癌生成、转移的免疫学机制提供新的思路,寻找免疫干预肝癌转移的方法。 进一步研究经负载不同转移潜能肝癌抗原的树突状细胞(DC)诱导的激活T细胞免疫功能重建对小鼠高、低淋巴结转移潜能的肝癌的影响,同时进一步研究影响肝癌转移的免疫学因素,为肝癌生成、转移的免疫学机制。 探讨小鼠淋巴道高、低转移潜能的肝癌细胞在体内淋巴道转移状况和淋巴管生成对淋巴道转移的影响并分析原因。 研究方法 分离Balb/c小鼠的T淋巴细胞,尾静脉注射入接种高、低转移潜能小鼠肝癌细胞株的Balb/c nu/nu裸鼠体内,每周重复注射一次,建立免疫功能重建模型。通过检测实验裸鼠体内的淋巴细胞比例和淋巴细胞功能评定模型的效果。 对T细胞免疫功能重建后的裸鼠足垫接种淋巴道高、低转移潜能细胞株,观察足垫接种小鼠不同转移潜能肿瘤的生长速度,转移程度。进一步应用DNA微阵列技术对免疫重建后的裸鼠体内的淋巴细胞进行免疫相关基因的检测。应用流式细胞仪对不同转移潜能的肿瘤细胞表面的MHC-Ⅰ、MHC-Ⅱ进行检测。应用半定量RT-PCR对肿瘤细胞分泌的免疫抑制相关因子IL-10、TGFα、TGFβ的程度进行进一步检测,寻找影响肝癌转移的免疫学原因。 应用激活的T淋巴细胞进行免疫功能重建。采用Balb/c小鼠的骨髓干细胞在IL-4与GM-CSF诱导下分化成DC,再负载淋巴道高、低转移潜能的小鼠肝癌抗原,诱导Balb/c小鼠T淋巴细胞,对Balb/c nu/nu小鼠进行免疫重建,观察足垫接种小鼠不同转移潜能肿瘤的生长速度,转移程度。应用双向混合淋巴细胞反应体外检测免疫重建的效果。用淋巴细胞增值实验检测肿瘤细胞对淋巴细胞的免疫抑制作用。采用ELISA法检测免疫重建前后裸鼠体内免疫抑制相关因子IL-10、TGF以及血管/淋巴管生成相关因因子VEGF的变化。应用Tunel检测方法对免疫重建组及未重建组的小鼠肿瘤凋亡进行进一步的检测。应用四步离心法分离高低转移潜能肝癌细胞株的Exosomes,进行电镜检测,并应用更多还原

【Abstract】 AIM: To establish a reconstructive model of T lymphocytes function and offer a platform to study the relationship between metastasis of hepatic carcinoma and immune system of the body. Then this model was used to study the effect of high and low metastasis to lymph nodes in mice and was also used to study the immune factors of the metastasis of hepatic carcinoma in order to find a new way to affect the metastasis of tumor by immune method and order a new method of immunotherapy.The further study is to use the T lymphocytes induced by dendritic cells loaded with corresponding hepatic carcinoma antigen to reconstruct the mice. The effct to lymph nodes metastasis of this method was studied.We also try to find out the relationship between lymphangiogenesis and lymph node metastasis in mice loaded with hepatic carcinoma in food pad. The reason of lymph node metastasis was studied.Methods: T lymphocytes of Balb/c mice was isolated and injected into Balb/c nu/nu mice with high and low metastasis hepatic carcinoma by caudal vein. This method was repeated every one week in next three weeks. Then the ratio of the distribution of CD3, CD4, CD8, CD4/CD8 in peripheral blood and spleen and the function of T lymphocytes was detected. This method was used to evaluate the effect of this model.Then the high and low metastases of lymph nodes cell line were inoculated into nude mice and the tumor’s growth and metastasis rate was observed. The lymphocytes’immune-associated gene was detected by cDNA micro array. The MHC-I and MHC- II of the tumor cells were detected by flow cytometer. The immunosuppression associated factor IL-10, TGF a and TGFβ secreted by high and low potential metastasis hepetic carcinoma was detected by demi-quantitative RT-PCR in order to find the immune reason of hepatic carcinoma metastasis to lymph nodes.We then use the activated T lymphocytes reconstruction to observe the effect of the metastasis of hepatic carcinoma. The bone stem cells of Balb/c mice were isolated and induced by IL-4 and GM-CSF to differentiate into dendritic cells (DC). The DCs were loaded with corresponding high and low metastasis potential hepatic cell antigen and then to induce the T lymphocytes of Balb/c mice into activated T lymphocytes. Balb/c nu/nu mice were distributed into 4 groups as immune reconstruction groups and no immune reconstruction groups of high and low metastasis hepatic carcinoma.The activated T lymphocytes were reconstructed into the Balb/c nude mice loaded with high and low metastasis potential carcinoma. The tumor’s growth and potential of metastasis rate of each group was observed. The effect of immune reconstruction was detected by di-mixture lymphocytes response in vitro. The immunosuppressive action was detected by lymphocyte value increase experiment of high and low potential metastasis tumor cells’ supernatant. The change of immunosuppression factors such as IL-10, TGF and VEGF were detected by ELISA. The apoptosis of high and low metastasis potential hepatic carcinoma after and before immune reconstruction of mice’ footpads were detected by TUNEL. The exosomes of high and low metastasis potential hepatic carcinoma was isolated by four-step centrifuging and was observed by electron microscope. The protic analysis was performed by SELDI-TOF-MS (Serface Enhanced Laser Desorption Inhibition Time Of Flight Ionization Mass Spectrometry) , in the exosomes of high and low metastasis potential hepatic carcinoma. The kill of tumor cell experiment was performed by T lymphocytes activated by DC loaded with exosomes of corresponding high and low metastasis potential hepatic carcinoma.Balb/c mice were distributed into two groups as high and low metastasis ones. The high and low potential metastasis to lymph node of hepatic carcinoma cell lines were injected into the footpad of Balb/c mice and the formation of tumor was observed. The lymph nodes of popliteal, inguinal region, para-common iliac a. and renal hilar were obtained to observed the metastasis potential of each group. The tumor constitution of footpad of each group was resected and made into cryo-section preparatio to perform 5’-nucleotidase-ALP double staining to observe the lymphoangiogenesis. Then high and low metastasis potential hepatic carcinomas were injected into the abdominal cavity of 615 mice to to form ascitic fluid. The ascitic fluid was get to perform lymphoangiogenesis experiment in vitro. The quantity of lymphatic vessels was calculated every 3 days. The total RNA of high and low metastasis potential cell line was extracted to perform metastasis gene DNA array examination. The VEGF-C, VEGF-D of each cell lines was detected by demi-quantitative RT-PCR and was furthur quantu analysis by real time PCR.RESULTS: The CD3 of T lymphocytes in peripheral blood and spleen, CD4 and CD4/CD8 of T lymphocytes in spleen of nude mice after immune reconstructed were significant higher than the control group (/K0.05) . The T lymphocytes of high metastasis potential can kill the corresponding tumor cell significantly (p<0.05).The growth rate of hepatic carcinoma in footpad of mice after immune reconstruction is lower than the control group and in high metastasis group is higher than low metastasis group. The metastasis to para-common iliac a. and renal hilar lymph nodes in immune reconstruction group is significant lower than the control group (p<0.05). The change of Thl/Th2 lymphocyte is occurrent in nude mice after immune reconstruction by cDNA micro array. The secretion of immunosuppression factors of tumor cell lines was detected by demi-quantitative RT-PCR. The MHC-1 in high metastasis group is significant higher than low metastasis group(p<.0.05), but the expression of MHC- II in each group have no significant difference.The growth rate of tumor after immune reconstruction by active T lymphocytes is significant lower than the control group(p<0.05). And the growth rate of tumor in high metastasis group is higher than low group. The metastasis potency to para-common iliac a. and renal hilar lymph nodes is lower than the control group after immune reconstruction (p<0.05). The supertanant of high and low metastasis tumor cell can significant improve the growth of the lymphocytes (p<0.05). The level of IL-10 in the serum of nude mice after immune reconstruction is significant than the control group (p<0.05). The VEGF level in serum of high metastasis group is higher than control group and in low metastasis group is lower than control group after immune reconstruction (p<0.05). The secret of TGF in nude mice after immune reconstruction has no significant difference. The killing effect of T cells induced by DC loaded with exosomes isolated by high and low hepatic carcinoma has no significant deference (p>0.05).The average time of tumor formation in Balb/c footpad is 11.5 +1.3 days. The metastasis to lymph nodes of popliteal and inguinal region has no significant difference in each group (p>0.05). But the metastasis to lymph nods of para-common iliac a. and renal hilar has significant difference (p>0.05). The quantity of lymphatic vessels in high metastasis group is significant higher than control group (p<0.05), but in low metastasis group has no significant difference. The expression of CD44n E-cadherin> Neu/HER2> H-Ras> VEGF-C in high metastasis group were higher and nm23A^ nm23-E4, pl6ink4a> CD61 were lower than low metastasis group in cDNA micro array experiment. And the VEGF-C in high metastasis group is higher and VEGF-D is lower than low metastasis group in demi-quantitative RT-PCR experiment. The secret of VEGF-D and VEGF-C/VEGF-D is significant lower in high metastasis group is significant lower than low metastasis group and VEGF-C has no significant更多还原