【作者】 张鹏；

【导师】 宋来君；

【作者基本信息】 郑州大学 ， 神经外科， 2011， 博士

【摘要】 脑胶质瘤,亦称为神经胶质细胞瘤,发生于神经外胚层,是颅内最常见的原发性肿瘤,约占全部颅内原发肿瘤的36.26%～60.96%。脑胶质瘤尤其是高级别胶质瘤具有异常增生、浸润性生长、易复发、血管异常增生及免疫耐受等生物学特性,这使得胶质瘤虽经不断改进的显微外科手术切除、放射治疗、化学治疗等措施的综合处理,仍易复发,预后不尽人意。如胶质母细胞瘤(Glioblastoma, GBM)患者虽经过综合治疗后,其中位生存时间仍少于15个月。为此临床需要寻找治疗胶质瘤的新方法。最近的研究证实,同种异体NK细胞能有效地杀伤原代的鼻咽癌、胃癌、肾癌、肠癌和卵巢癌等肿瘤细胞。NK细胞对靶细胞的杀伤活性与NK细胞表面的受体和靶细胞表面的配体之间的相互作用密切相关。NK细胞表面的杀伤抑制受体与靶细胞表面的HLA-I类分子的结合,抑制NK细胞对靶细胞的杀伤,而当靶细胞缺乏HLA-I类分子时可激发NK细胞对靶细胞的杀伤作用。NKG2D(natural killer group 2D)是NK细胞表面的主要杀伤活化受体,其配体为MHC-Ⅰ类链相关分子(MHC classⅠchain-related molecule, MIC) A和B及人巨细胞病毒UL16蛋白的结合蛋白(UL16 binding proteins, ULBP)1、2和3等。本研究关注的是同种异体的NK细胞是否具有杀伤胶质瘤细胞的效应。在体外条件下分离、培养人脑胶质瘤细胞(human glioma cell, HGC),观察其生物学特性,检测其HLA-Ⅰ类分子和NKG2D配体的表达,并在体内、外条件下观察同种异体NK细胞杀伤胶质瘤细胞的可行性及其分子基础,为进一步认识同种异体NK细胞对胶质瘤细胞的杀伤作用提供实验和理论基础。从而为胶质瘤的治疗探索新的方法。第一部分原代培养HGC的生物学特性研究目的：原代分离、培养、鉴定HGC,并观察其生物学特性,为探讨同种异体NK细胞对HGC的杀伤效应提供必备的基础。方法：术中获取胶质瘤患者肿瘤组织标本(术后病理诊断为胶质瘤Ⅳ级),分离、培养得到HGC,并经GFAP免疫组织化学鉴定为HGC。镜下观察胶质瘤细胞生长特点,并将一定剂量的HGC注入BALB/c裸鼠体内,观察移植HGC成瘤特征。结果：1.显微镜下观察肿瘤组织HE染色切片见：核浓染,异形性明显,可见巨核,核分裂等。判定培养细胞来源的组织为胶质瘤Ⅳ级。2.体外培养的HGC生长良好,群体倍增时间为23.48hr。传代后的细胞生长旺盛,状态良好,性状稳定,有明显的对数生长期。3.培养的HGC经HE染色显示：细胞呈梭形、星形或不规则形,突起较小,核大小不一。免疫细胞化学结果显示：绝大多数细胞GFAP呈阳性反应。4.将0.1ml (1×106个) HGC的悬液移植入BALB/c裸鼠体内,肿瘤出现时间为(9.17±1.68)d,成瘤率为100%,瘤组织为胶质瘤Ⅳ级。结论：1.HGC可以在体外进行培养,细胞倍增时间为23.48hr。2.胶质瘤细胞可成瘤,肿瘤出现时间约为(9.17±1.68)d。第二部分HGC HLA-I类分子和NKG2D配体表达的检测目的：检测原代培养的HGC HLA-I类分子和NKG2D配体表达情况,为进一步认识同种异体NK细胞对胶质瘤细胞的杀伤作用提供实验和理论基础。方法：采用RT-PCR的方法检测HGC MICA、MICB、ULBP1、ULBP2及ULBP3在mRNA水平的表达；采用流式细胞技术检测MICA、MICB、ULBP1、ULBP2、ULBP3分子及HLA-I类分子的表达；采用Western Blot技术检测MICA.ULBP2及ULBP3蛋白的表达。结果：1. RT-PCR检测结果显示HGC在mRNA水平表达NKG2D的配体MICA、MICB、ULBP1、ULBP2和ULBP3。2.流式细胞仪检测结果证实HGC表面表达HLA-I类分子及NKG2D的配体MICA、ULBP2和ULBP3,不表达NKG2D的配体MICB及ULBP1。3. MICA、ULBP2、ULBP3和HLA-I类分子在HGC的表达的阳性百分数分别为63.26±6.71%、61.234±5.97%、51.47±2.43%和89.67±7.28%。4. Western blot检测结果证实胶质瘤细胞表而表达NKG2D的配体MICA、ULBP2和ULBP3分子。结论：1.HGC在mRNA水平表达NKG2D的配体ULBP1、ULBP2、ULBP3、MICA和MICB。2.HGC表面表达MICA、ULBP2及ULBP3分子,不表达MICB和ULBP1分子。3.HGC表面高表达HLA-I类分子。第三部分同种异体NK细胞对HGC体外杀伤效应的实验研究目的：检测同种异体NK细胞对HGC的杀伤效应。方法：采用密度梯度离心法分离外周血单个核细胞,并通过MACS纯化NK细胞；采用MTT方法检测同种异体NK细胞对HGC杀伤效应,及不同效靶比时NK细胞对HGC的杀伤活性。结果：1.分离、纯化的阳性部分中,CD3-CD16+CD56+细胞的纯度达91.7%。2.同种异体NK细胞对HGC具有杀伤活性,且杀伤效应随着效靶比的升高而增强；效靶比为10∶1、20∶1、50∶1和100∶1时,杀伤活性分别为：23.64±1.73%、37.28±3.67%、49.63±4.28%和74.16±6.43%。结论：1同种异体NK细胞对HGC具有杀伤活性。2 HLA-I类分子及MICA、ULBP2及ULBP3分子在NK细胞对HGC的杀伤效应中起重要作用。第四部分同种异体NK细胞对HGC裸鼠皮下移植瘤的抑制作用目的：观察同种异体NK细胞在BALB/c裸鼠体内对HGC的杀伤效果,为脑胶质瘤免疫治疗提供实验和理论基础。方法：BALB/c裸鼠32只,随机分为4组：A组：无细胞移植组；B组：HGC移植组；C组：HGC+NK细胞移植治疗组；D组：单纯NK细胞移植组,每组8只。观察各组裸鼠成瘤时间、肿瘤体积变化,绘制肿瘤生长曲线、计算成瘤率。成瘤3周后,处死裸鼠,取瘤块称重,计算肿瘤生长抑制率,肿瘤组织常规病理检查。结果：1.A组和D组无肿瘤生长,B组和C组肿瘤出现时间分别为(9.23±1.64)d和(15.26±1.53)d。2.生长曲线显示同种异体NK细胞能明显减缓移植入BALB/c裸鼠体内的HGC的生长速度。B组和C组裸鼠的瘤体重量分别为(1.74±0.13)g和(1.26±0.08)g,C组瘤体较B组瘤体明显减小,肿瘤生长抑制率为27.59%。3.成瘤组织经HE染色鉴定为胶质瘤Ⅳ级,C组瘤体HE染色切片可见较多的坏死肿瘤细胞和NK细胞浸润。结论：同种异体NK细胞能有效抑制移植到裸鼠体内的HGC的生长。更多还原

【Abstract】 Glioma, also known as neurogliocytoma, is originated from neural ectoderm. It is the most common primary intracranial tumor, accounting for about 36.26%～60.96% of all primary intracranial tumors. Glioma, especially high-grade glioma, is characterized by abnormal proliferation, infiltrative growth, tendency to relapse, abnormally vascular hyperplasia and immune tolerance. Therefore, even treated with comprehensive measures including constantly improved microsurgical resection, radiotherapy and chemotherapy, glioma is still easy to relapse with undesirable prognosis. For example, though treated with comprehensive therapy, the median survival time of glioblastoma (GBM) is still less than 15 months. So new methods for the treatment of glioma should be explored in clinical practice.Recent research has proved that allogeneic NK cells can effectively kill cells of nasopharyngeal carcinoma, gastric carcinoma, renal carcinoma, intestinal cancer and ovarian cancer. The cytotoxicity of NK cells against target cells is closely related to receptors on the surface of NK cells and ligands on the surface of target cells. Killer receptors on the surface of NK cells bind to HLA class I molecules on the surface of target cells, inhibiting the cytotoxicity of NK cells against target cells. When target cells lack HLA classⅠmolecules, NK cells are activated to kill target cells. NKG2D (natural killer group 2D) is the main killer activatory receptor on the surface of NK cells. Its ligands are MHC classⅠchain-related molecules (MIC) A and B and human cytomegalovirus UL16 binding proteins (ULBP) 1,2 and 3.What this study focuses on is whether allogeneic NK cells have cytotoxic effect on glioma cells or not. In this study, we isolated and cultured human glioma cells in vitro, observed their biological characteristics, detected the expression of HLA class I molecules and NKG2D ligands on human glioma cells, and observed the cytotoxicity of allogeneic NK cells against glioma cells and its molecular basis in vivo and in vitro, providing experimental and theoretical basis to further understand the feasibility and mechanism of cytotoxicity of allogeneic NK cells against glioma cells. Thus we explored a new method for the treatment of glioma.PartⅠ. Biological characteristics of primary cultured human glioma cellsObjective:To provide the necessary basis to explore cytotoxicity of allogeneic NK cells against human glioma cells by primary culture and identification human glioma cells and observe their biological property.Methods:Human glioma cells were isolated from tissue samples obtained during the surgery of patients who were clinically and pathologically diagnosed with glioma gradeⅣ, and the cells were primarily cultured and identified as human glioma cells by immunohistochemistry assay of glial fibrillary acidic portein (GFAP).The growth characteristics of glioma cells was observed under microscope. The growth curve was analyzed by MTT method. Human glioma cells of a certain amount were transplanted into BALB/c nude mice. Time of tumor formation, tumor formation rate and tumor growth curve were observed. Results:1. It was observed under tumor tissue HE stained slices that the nuclei were heavily stained with significant heteromorphism and that macronuclei and nuclear division were visible. The source tissue of cell culture was identified as glioma gradeⅣ.2. Human glioma cells cultured in vitro grew well, with a population density doubling time of 23.48 hr. Subcultured cells grew vigorously in a good status. The property was stable with an obvious exponential phase of growth.3. HE staining of cultured human glioma cells showed that the cells were in fusiform, starlike or irregular shapes with small prominences and with nucleus of variable sizes. Results of immunocytochemistry showed that most cells were positive for GFAP staining. There were yellow or golden brown positive substances of various concentrations in cytoplasm.4. The time of tumor formation was (9.17±1.68)d days after transplanting 0.1ml (1×106) suspension of glioma cells into BALB/c nude mice. Tumor formation rate was 100%. Tumor tissue was pathologically diagnosed as glioma gradeⅣ.Conclusions:1. Glioma cells can be cultured in vitro, and cell doubling time was 23.48 hr.2. Glioma cells can grow to form tumor. The time of tumor formation was about (9.17±1.68) days. Tumor grew rapidly with obvious pathological morphology.PartⅡ. Detection of the expression of HLA ClassⅠmolecules and NKG2D ligands in human glioma cellsObjective:To detect the expression of HLA classⅠmolecules and NKG2D ligands in primary culture human glioma cells, providing experimental and theoretical basis to further understand cytotoxicity of allogeneic NK cells against glioma cells.Methods:Reverse transcription PCR method was used to detect the expression of MICA, MICB, ULBP1, ULBP2 and ULBP3 in human glioma cells at mRNA level. Flow cytometry was used to detect the expression of MICA, MICB, ULBP1, ULBP2, ULBP3 and HLA classⅠmolecules. Western Blot technique was used to detect the expression of MICA, ULBP2 and ULBP3 proteins.Results:1. Results of RT-PCR showed that MICA, MICB, ULBP1, ULBP2 and ULBP3 were all expressed in human glioma cells at mRNA level.2. Results of flow cytometry confirmed NKG2D ligands MICA, ULBP2 and ULBP3 were expressed on the surface of human glioma cells while NKG2D ligands MICB and ULBP1 were not expressed on the surface of human glioma cells.3. The expression percentage of MICA, ULBP2, ULBP3 and HLA classⅠmolecules in human glioma cells were 63.26±6.71%,61.23±5.97%,51.47±2.43% and 89.67±7.28% respectively; Results of Western Blot confirmed that NKG2D ligands MICA, ULBP2 and ULBP3 proteins were expressed in glioma cells.Conclusions:1. NKG2D ligands ULBP1, ULBP2, ULBP3, MICA and MICB were expressed in human glioma cells at mRNA level.2. MICA, ULBP2 and ULBP3 are expressed on the surface of human glioma cells while MICB and ULBP1 are not expressed on the surface of human glioma cells.3. HLA classⅠmolecules are highly expressed on the surface of human glioma cells. PartⅢ. Study of cytotoxicity of allogeneic NK cells against human glioma cells in vitroObjective:To detect cytotoxicity of allogeneic NK cells against human glioma cells.Methods:Density gradient centrifugation was used to isolate mononuclear cells from peripheral blood and NK cells were purified through MACS; MTT method was used to detect cytotoxicity of NK cells against human glioma cells and cytotoxic activity of different effector-target ratios.Results:1. Among the isolated and purified positive cells, the proportion of CD3-CD 16+ CD56+ cells was 91.7%.2. Allogeneic NK cells showed cytotoxic activity against human glioma cells, which increased with the rise of effector-target ratio; when effector-target ratio was 5:1,20:1,50:1 and 100:1, the toxic activity was 23.64±1.73%,37.28±3.67%, 49.63±4.28% and 74.16±6.43% respectively.Conclusions:1. Human allogeneic NK cells have cytotoxic activity against glioma cells.2. HLA class I molecules, MICA, ULBP2 and ULBP3 play important roles in the cytotoxic effect of NK cells on glioma cells.PartⅣ. Inhibitory effect of allogeneic NK cells on human glioma cells in nude miceObjective:To provide experimental and theoretical basis for immunotherapy of glioma by observing cytotoxicity of allogeneic NK cells against human glioma cells in BLAB/c nude mice,Methods:32 BALB/c nude mice were randomly divided into 4 groups:group A: without cell transplantation; group B:Human glioma cells transplantation; group C: Human glioma cells+NK cells treatment; group D:NK cells transplantation, eight mice in each group. The time of tumor formation, tumor formation rate and changes of tumor size were observed for nude mice in each group, and tumor growth curves were drawn. Three weeks after tumor formation, mice were euthanized. Tumors were removed and weighted. Tumor inhibition rates were calculated and routine pathological examination was performed for tumor tissues.Results:1. There was no tumor in A and D group. In group B and group C, the times of tumor formation was (9.23±1.64) days and (15.26±1.53) days respectively.2. Growth curve showed NK cells could significantly slow down the growth of transplanted tumor from glioma cells in BALB/c nude mice. In group B and group C, the tumor masses were (1.74±0.13) g and (1.26±0.08) g respectively. Tumor size in group C was significantly smaller than that in group B. Tumor inhibition rate of group C was 27.59%.3. Through HE stainin, the specimen of tumors from nude mice were identified as glioma gradeⅣ. Under microscope, there were many necrotic tumor cells and NK cell infiltration on HE stained slices of tumor in group C.Conclusion:The human allogeneic NK cells can effectively inhibit the growth of human glioma cells which were transplanted in BALB/c nude mice.更多还原