發(fā)布時(shí)間：2018-08-28 16:59

【摘要】：目的 腎細(xì)胞癌(Renal cell carcinoma, RCC)占男性新發(fā)癌癥的4%,在女性新發(fā)癌癥病例中占3%,與其他實(shí)體腫瘤不同的是,腎癌對(duì)腫瘤的傳統(tǒng)療法均不敏感,如放射治療、化學(xué)治療等.但是腎癌被發(fā)現(xiàn)是免疫相關(guān)的腫瘤,多種免疫治療在腎癌的治療中取得了不同程度的成功。本實(shí)驗(yàn)通過(guò)研究過(guò)繼細(xì)胞免疫治療(Adoptive cellular immunotherapy, ACI):異體抗CD3分子的單克隆抗體激活的殺傷細(xì)胞(anti-CD3monoclonal antibody activated killer cells, CD3AK)對(duì)人腎癌細(xì)胞株OS-RC-2的體外殺傷作用,以及其對(duì)嚴(yán)重聯(lián)合免疫缺陷(severe combined immunodeficiency, SCID)小鼠人腎癌移植瘤模型的體內(nèi)抑制作用,從而進(jìn)一步探討異體CD3AK細(xì)胞對(duì)SCID小鼠人腎癌移植瘤治療的有效性,為異體CD3AK細(xì)胞治療腎癌的臨床應(yīng)用提供理論及實(shí)驗(yàn)支持。 方法 1.人腎癌OS-RC-2細(xì)胞株的培養(yǎng)與傳代：無(wú)菌細(xì)胞培養(yǎng),及時(shí)進(jìn)行換液與傳代,定時(shí)觀察細(xì)胞形態(tài)與數(shù)量,待細(xì)胞進(jìn)入對(duì)數(shù)生長(zhǎng)期時(shí),臺(tái)盼藍(lán)染色鑒定活力后用于實(shí)驗(yàn)。 2.于體外條件下建立大量擴(kuò)增異體CD3AK細(xì)胞的培養(yǎng)體系：采集健康志愿者的外周血,體外分離獲得外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cell, PBMC),培養(yǎng)獲得異體CD3AK細(xì)胞；采用流式細(xì)胞儀檢測(cè)CD3AK細(xì)胞的表型； 3.CCK-8(Cell Counting Kit-8)法檢測(cè)異體CD3AK細(xì)胞對(duì)人腎癌0S-RC-2細(xì)胞株的殺傷作用； 4.建立荷瘤模型：采用左側(cè)腹股溝皮下注射的方法,建立人腎癌SCID鼠皮下移植瘤模型,并通過(guò)尾靜脈注射異體CD3AK細(xì)胞,對(duì)照組尾靜脈注射磷酸鹽緩沖液(phosphate buffer solution, PBS),劑量為0.2m1／只,觀察小鼠的生物學(xué)特征。SCID小鼠被隨機(jī)分為四組(n=8)：A組(于荷瘤前尾靜脈注射異體CD3AK細(xì)胞),B組(A組對(duì)照組：荷瘤前尾靜脈注射PBS)；C組(荷瘤后尾靜脈注射異體CD3AK細(xì)胞),D組(C組對(duì)照組：荷瘤后尾靜脈注射PBS)。隔日測(cè)量小鼠的體重、腫瘤體積以觀察小鼠體重及腫瘤生長(zhǎng)變化；末次治療結(jié)束后24小時(shí)處死小鼠,無(wú)菌取脾臟、腫瘤組織用于后續(xù)實(shí)驗(yàn)。 5.免疫組化法檢測(cè)CD3+細(xì)胞在小鼠腫瘤組織中的表達(dá)情況；計(jì)算脾臟指數(shù)(脾重／體重),觀察異體CD3AK細(xì)胞對(duì)腎腫瘤生長(zhǎng)的抑制作用及其對(duì)小鼠免疫系統(tǒng)的作用。 結(jié)果 1.將異體CD3AK細(xì)胞與人腎癌OS-RC-2細(xì)胞共同孵育12h,用CCK-8法檢測(cè)可以觀察到,隨著效應(yīng)細(xì)胞與靶細(xì)胞比值的增大,異體CD3AK細(xì)胞對(duì)OS-RC-2細(xì)胞的殺傷活性隨之增高。 2.A、C組小鼠與其對(duì)照組B、D組小鼠比較,A、C組小鼠的脾臟指數(shù)顯著增大(P0.05)；A組與C組比較,小鼠脾臟指數(shù)無(wú)明顯差異(P0.05)； 3.A、C組小鼠與其對(duì)照組B、D組小鼠比較,A、C組小鼠腫瘤重量顯著減小,腫瘤生長(zhǎng)緩慢(P0.05)；A、C組與B、D組小鼠體重均增加,實(shí)驗(yàn)(A、C)組小鼠體重較其對(duì)照(B、D)組小鼠體重增加的少(P0.05), 4.A組小鼠與C組小鼠比較,A組小鼠腫瘤重量減小,腫瘤生長(zhǎng)遲緩(P0.05)。 5.免疫組化方法示,CD3+細(xì)胞在C組小鼠腫瘤組織中有陽(yáng)性表達(dá),其余各組小鼠腫瘤組織未發(fā)現(xiàn)有陽(yáng)性表達(dá)。 結(jié)論 1.異體CD3AK細(xì)胞對(duì)人腎癌OS-RC-2細(xì)胞株具有體外殺傷作用； 2.異體CD3AK細(xì)胞對(duì)人腎癌SCID小鼠移植瘤的生長(zhǎng)具有顯著抑制作用； 3.異體CD3AK細(xì)胞對(duì)人腎癌SCID小鼠移植瘤的發(fā)生具有一定的預(yù)防作用； 4.異體CD3AK細(xì)胞能增強(qiáng)人腎癌荷瘤SCID小鼠機(jī)體免疫功能并發(fā)揮其抗腫瘤作用。
[Abstract]:objective
Renal cell carcinoma (RCC) accounts for 4% of all new cancers in men and 3% of all new cancers in women. Unlike other solid tumors, RCC is insensitive to traditional therapies such as radiotherapy and chemotherapy. In this study, we investigated the in vitro killing effect of anti-CD3 monoclonal antibody activated killer cells (CD3AK) on human renal cell carcinoma cell line OS-RC-2 and its severe association with adoptive cell immunotherapy (ACI). The inhibitory effect of allogeneic CD3AK cells on human renal cell carcinoma xenograft tumor in SCID mice was investigated in vivo, which provided theoretical and experimental support for the clinical application of allogeneic CD3AK cells in the treatment of renal cell carcinoma.
Method
1. Culture and passage of human renal cell carcinoma OS-RC-2 cell line: aseptic cell culture, timely liquid exchange and passage, regular observation of cell morphology and quantity, when the cell entered the logarithmic growth phase, trypan blue staining identified the viability of the experiment.
2. Establish a culture system of CD3AK cells in vitro. Collect peripheral blood mononuclear cells (PBMC) from healthy volunteers and isolate PBMC in vitro to obtain allogeneic CD3AK cells. Flow cytometry was used to detect the phenotype of CD3AK cells.
3.CCK-8 (Cell Counting Kit-8) method was used to detect the killing effect of CD3AK cells on human renal cell carcinoma 0S-RC-2 cell line.
4. Establishment of tumor-bearing model: SCID mice were subcutaneously injected into the left groin to establish a model of human renal cell carcinoma. Allogeneic CD3AK cells were injected into the tail vein, and phosphate buffer solution (PBS) was injected into the tail vein of the control group. The biological characteristics of the mice were observed at a dose of 0.2m1/mouse. They were divided into four groups (n=8): group A (injecting allogeneic CD3AK cells into the anterior and caudal veins of tumor-bearing mice), group B (control group A: injecting PBS into the anterior and caudal veins of tumor-bearing mice), group C (injecting allogeneic CD3AK cells into the posterior tail veins of tumor-bearing mice) and group D (control group C: injecting PBS into the tail veins of tumor-bearing mice). The mice were sacrificed 24 hours after the end of the last treatment, and the spleen was removed by asepsis. The tumor tissues were used for subsequent experiments.
5. Immunohistochemistry was used to detect the expression of CD3+ cells in tumor tissues of mice, and spleen index (spleen weight/body weight) was calculated to observe the inhibitory effect of allogeneic CD3AK cells on the growth of renal tumor and the immune system of mice.
Result
1. Allogeneic CD3AK cells were incubated with human renal cell carcinoma OS-RC-2 cells for 12 hours. CCK-8 assay showed that the cytotoxicity of allogeneic CD3AK cells to OS-RC-2 cells increased with the increase of the ratio of effector cells to target cells.
2. The spleen index of mice in group A and C was significantly higher than that of mice in group B and D (P 0.05), and there was no significant difference between group A and group C (P 0.05).
3. Compared with the control group B, D mice, the weight of tumors in group A, C decreased significantly and the growth of tumors was slow (P 0.05); The weight of mice in group A, C and B, D increased significantly, and the weight of mice in group A and C increased less than that of mice in group B and D (P 0.05).
Compared with group C, mice in group 4.A showed less tumor weight and slower tumor growth (P0.05) than group A mice.
5. Immunohistochemical staining showed that CD3+ cells were positively expressed in tumor tissues of mice in group C, but no positive expression was found in tumor tissues of mice in other groups.
conclusion
1. allogeneic CD3AK cells have an in vitro killing effect on human renal cell carcinoma OS-RC-2 cell line.
2. allogeneic CD3AK cells significantly inhibited the growth of transplanted human renal cell carcinoma SCID mice.
3. allogeneic CD3AK cells have a certain preventive effect on the transplanted tumor of human renal carcinoma SCID mice.
4. allogeneic CD3AK cells can enhance the immune function of human renal cell carcinoma bearing SCID mice and play an anti-tumor role.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.11

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