摘要
卵巢癌是女性生殖器官三大恶性肿瘤之一,死亡率占据妇科恶性肿瘤首位,预后差。目前已有的卵巢癌肿瘤治疗方法包括手术治疗,化学药物治疗和放射治疗,但是都有其局限性。从而,迫使人们努力寻找新的治疗手段,以达到特异而有效的识别并杀灭肿瘤细胞的目的。其中生物学治疗手段已引起人们的广泛关注。生物治疗包括非特异的细胞因子(白介素-2,干扰素-α等)疗法和单克隆抗体、αβ细胞毒性T细胞(CTL)过继治疗等靶向治疗方法,对微小肿瘤以及残留病变的清除有一定的疗效。但是单克隆抗体和αβT阻细胞精密识别靶抗原的优点又限定了其抗肿瘤治疗的肿瘤谱,同时治疗用的大量抗体和CTLs的来源也是不易解决的问题。目前,γδT细胞因其识别抗原广泛,且识别无MHC分子限制性,成为过继治疗的候选细胞。但在γδT细胞制剂的制备方法上仍存在某些问题亟待解决。如中晚期肿瘤患者或化疗后患者体内免疫抑制,导致种子细胞数量减少和免疫活性下降,致使体外扩增细胞数量受限,难以达到治疗所需数量的细胞制剂。因此使用基因工程方法制备基因修饰的γδT淋巴细胞治疗肿瘤不失为一个值得尝试的新型策略。
近年来,我们实验室选择TCRδ链的CDR3 (CDR3δ)为研究对象,通过CDR3合成多肽、CDR3移植性Ig与肿瘤靶细胞、靶组织以及肿瘤细胞提取蛋白的相互作用的实验,验证了CDR3是TCRyδ与抗原结合的关键部位,进而证明γδ6T细胞能够经由其细胞受体(yδTCR)δ链的CDR3区(CDR3δ)广泛地识别、进而杀伤多种实体肿瘤细胞。
因此,本文的第一部分旨在利用基因工程技术,制备特异肿瘤反应性的CDR3δ移植型γδT细胞。通过体内体外实验以证明其对肿瘤具有杀伤作用,从而为临床过继性免疫治疗提供新思路和新方法。
本研究主要包括以下几个方面的工作：
一、建立mRNA电转外周血淋巴细胞(PBLs)技术的平台用于移植的肿瘤特异性的CDR3δ序列的筛选。
将线性化的pGEM4Z/EGFP/A64质粒作为体外转录RNA的模板,通过不同的电转参数下细胞电转率及细胞存活率的比较,建立mRNA电转的平台。将实验室前期从卵巢癌TIL获得的γδTCRδ2的特异性CDR3序列OT1,OT3和OT10分别嵌合进入全长的γδTCRδ2链,全长的γδTCRδ2链与γ9链分别体外转录成mRNA后,共同转染抗CD3抗体刺激的正常人PBMC,使其细胞膜表面表达TCRγδ,分别命名为γ9δ2(OTl)T细胞,γγ9δ2(OT3)T细胞和γ9δ2(T10)T细胞。检测上述转染细胞,在肿瘤细胞蛋白的刺激下,活化分泌的细胞因子及其对多种肿瘤细胞的细胞毒作用。结果显示,相对于γ9δ2(OT1)T细胞,γ9δ2(OT3)T细胞和γ9δ2(T10)T细胞具有更显著的细胞毒作用,且分泌更多的细胞因子。进而,结合实验室以前的研究结果,我们选用嵌合OT3序列的δ链和γ9链进行下一步的实验。
二、基因修饰的γ6 T淋巴细胞抑癌作用及其杀伤机制的研究
鉴于mRNA电转技术得到的基因修饰的淋巴细胞的转染效率低,表达时间短的缺陷,我们运用逆转录病毒技术制备基因修饰的淋巴细胞。我们分别包装含有OT3序列全长的γδTCRδ2链与γδTCRγ9链的逆转录病毒颗粒,将获得的高滴度病毒浓缩后,感染经抗CD3抗体刺激的正常人PBMC,使其细胞膜表面表达TCRγδ,命名为γ9δ2(OT3)T细胞。结果显示γγ9δ2(OT3)T细胞被多种肿瘤细胞蛋白刺激后,TNF-a和IFN-γγ分泌增加。同时,对多种肿瘤细胞也具有细胞毒作用。而且,这种细胞毒作用能被抗TCRγδ的单克隆抗体所阻断。这些结果提示,γ9δ2(OT3)T细胞能够被肿瘤抗原活化,且具有抗肿瘤作用。为了研究转染细胞细胞毒活性的作用机制,我们进行了Fas/FasL途径的抑制剂BFA和穿孔素／颗粒酶途径的抑制剂CMA对γγ9δ2(OT3)T细胞的细胞毒阻断实验。结果发现,BFA对γγ9δ2(OT3)T细胞肿瘤杀伤作用的最大抑制率仅有20-30%；CMA对γ9δ2(OT3)T杀伤Fas低表达的SKOV3细胞的杀伤活性抑制率可达56.71%,对Fas高表达的H08910细胞的杀伤活性抑制率达33.93%。联合应用CMA和BFA对γγ9δ2(OT3)T细胞的细胞毒作用抑制率达60%以上。以上结果提示,γγ9δ2(OT3)T细胞对肿瘤的细胞毒作用中,穿孔素／颗粒酶和Fas/FasL途径都发挥作用,但穿孔素／颗粒酶途径起主要作用,尤其在对Fas低表达的肿瘤细胞的细胞毒作用中更为重要。
三、γ9δ2(OT3)T细胞体内抑癌作用研究
通过裸鼠荷人卵巢癌肿瘤模型,肿瘤局部注射γ9δ2(OT3)T细胞,观察其对肿瘤的治疗效果。结果显示,γ9δ2(OT3)T细胞配合IL-2治疗,相对于空载体感染的细胞配合IL-2治疗组,肿瘤的生长明显受到抑制,裸鼠的生存期也有所延长。
总之,本文第一部分通过将γδTCR基因序列转染进入抗CD3抗体刺激的外周血单个核细胞中,获得具有特异肿瘤反应性的免疫效应细胞。并且通过体内外实验验证了基因修饰细胞的抗肿瘤的能力,为肿瘤免疫治疗提供了新的思路与方法。
抑郁症的发病率和患病率较高,其严重影响了病人的生理、心理健康及社会交往,使病人的日常生活能力明显受损,增加了致残率和死亡率。传统理论认为,抑郁症主要是由于单胺神经递质类物质活性降低引起。最近,又有其他新的理论解释抑郁症的病理生理学。其中糖皮质激素,神经营养因子和免疫系统细胞因子网络的变化逐渐引起了人们的关注。目前对抑郁症的研究主要集中在单个细胞因子群,特别是单核促炎性细胞因子(IL-1β,IL-6,TNF-α)。然而,细胞因子之间具有多效性而且彼此之间密切相互作用。细胞因子属于不同的细胞群分泌对免疫反应有不同的应答。因此,本文的第二部分采用ELISA法研究对抑郁症患者血清中3个亚系的细胞因子,主要是单核细胞因子(IL-1β,IL-6,TNF-α),Thl细胞因子(IFN-γ和IL-2)及一个Th2细胞因子(IL-4)进行了研究。结果表明,抑郁症患者血清IL-1β和IL-6水平显著高于正常对照组,且血清IL-1与IL-6变化成正相关(r=0.308 P<0.01),提示IL-6与IL-1在抑郁症的发病过程中可能具有相关性。抑郁症患者TNF-α水平高于正常对照组,但是没有统计学差异。抑郁症患者血清中Thl型细胞因子水平明显低于正常对照组,Th2型细胞因子明显高于正常对照组。上述结果提示抑郁症患者Th1/Th2细胞因子分泌严重失衡。此外,我们用ELISA法检测了抑郁症患者血清中胶质细胞源性神经营养因子(GDNF)及MHCI类链相关基因A(MICA)的表达,结果发现,抑郁症患者血清GDNF显著低于对照组,MICA显著高于对照组。上述检测的指标在抑郁症病人接受6个星期的百优解及传统医学的针刺治疗后,都有明显的逆转。这提示GDNF和MICA可能是抑郁症的生物指标之一。然而其与抑郁症病因、病理变化机制及相关的神经递质的作用都需要进一步深入研究。总之,本文的第二部分初步研究了抑郁症患者外周血中细胞因子,GDNF及MICA的变化,为抑郁症的发病机制研究提供了一些依据,上述细胞因子有可能作为抑郁症患者临床诊断的辅助手段。
Ovarian cancer, one of the three malignant tumors in women, has the worst prognosis of all gynecological cancers and is a leading cause of death from cancer in women. Nowadays the routine treatments on ovarian cancer include surgery, chemotherapy and radiotherapy. Because of their limitations, more effective and specific ways in killing tumors are needed. Biological treatment has received a huge amount of attention in recent years. As a type of treatment that works with immune system,Biological treatments involving non-specific cytokines (IL-2, IFN-a) and target therapy of monoclonal antibodies and ap cytotoxic T cells (CTL) help to eliminate tiny tumors and remnant pathological changes. In contrast to non-specific cytokines, the great virtue of target therapy that monoclonal antibodies and ap T cells precisely recognize the tumor antigen restricts the application of monoclonal antibodies andαβT cells in clinical treatment. The acquirement of y8T cells ex vivo is a mature technology; however, there are still some problems. For example, the numbers and immunological activities of seed cells in the patients with median or late-stage malignant tumors or treated with chemotherapy decreased and are not up to quantities demand for treatment. Therefore, gene-modified y8 T lymphocytes consider being prepared for tumor therapy using genetic engineering methods.
In recent years, our lab selected the CDR3 of 82 chain (CDR38) as a research target and investigated the interaction of CDR3 peptide or CDR3 grafted Ig with tumor cells, tumor tissues or tumor cell protein extracts. Our results suggested that CDR3δwas the important position for antigen recognition and furthermoreγδT cells were able to widely recognize and kill many solid tumors through CDR3 of 8 chain.
In the first part of my thesis, we constructed gene modified y8 T cells whose CDR38 is replaced by sequences of OT3 and showed they had tumor specific reactivity and cytotoxity in vivo and in vitro. Our data indicated that the adoptive transfer of gene modified y8 T cells can provide a new strategy for cancer therapy.
The first part of the experiments contained the following work
1 Create technological platform for screen the tumor specific and reactive CDR3 region.
Linearized plasmid pGEM4Z/EGFP/A64 was template for in vitro mRNA. Compared the electroparation expression rates and cell survival rates under various parameters, we make sure the prior condition and build a platform of mRNA elctroparation. Stimulated PBMC were transferred by mRNA electroporation with the combination of the. full length ofδchain with different CDR3 region and the full length ofδchain from ovarian cancer infiltrated yδT cells and expressedγδTCR whose CDR3 region of 8 chain were replaced by OT1, OT3 or OT10 sequences, named y9δ2(OT1)T cells,γ9δ2(OT3)T cells and y9δ2(OT10)T cells separately. After stimulated by various tumor protein extracts,the transfectant cells expressing y8 TCR with specific CDR3 sequences secreted TNF-a, IFN-y. At the same time, transfentent cells showed the specific lysis against tumor cell lines. y9δ2(OT1)T cells are suboptimal for the cytotoxity and cytokines secretion in comparison to y9δ2(OT3)T cells and y982(OT10)T cells. Previous experiments demonstrated OT3 peptide has more affinity than OT10 peptide. Combined previous results of our laboratory, we chose the OT3 sequence as the CDR38 for the next studies.
2 Retro virus-mediated yδTCR gene transfer into PBLs and evaluation of their functional activity against tumors.
To improve the expression rate, we constructed TCRγ-andδ-chain gene retrovirus vectors. They were introduced into two individual vectors and generated TCR retrovirus vector particles. After 3 days activated, PBMC isolated from healthy volunteers were harvested, resuspended at 1 X 106 cells/ml inγδTCR retrovirus vector particles containing 200 IU/ml IL-2 and expressed yδTCR at the surface. The cells were called y982(OT3)T cells. After stimulated by various tumor protein extracts, the transfectant cells expressingγδTCR with OT3 sequence secreted much more TNF-a, IFN-y. At the same time,γ9δ2(OT3)T show the specific lysis against tumor cell lines and the lysis can be blocked by anti-γδTCR antibody. To study the mechanisms of the cytotoxocity, we incubated the y982(OT3)T cells with a FasL/Fas pathway inhibitor BFA or a perforin/granzyme inhibitor CMA at the different concentration before killing target cells. Results indicated that BFA partially blocked the killing ofγ9δ2(OT3)Tells against target cells (20-30%) and CMA suppressed the killing ability ofγ9δ2(OT3)T in Fashigh or Faslowcells, the inhibition rate was up to 33.93% and 56.71%, respectively. Combination CMA and BFA can mostly inhibit the killing process. These findings indicated that y982(OT3)T cells lysed their target cells via FasL/Fas and perforin/granzyme pathways, the latter was more important, especially in killing tumor cells that expressed low levels of Fas.
3 Tumor cell therapy with y982(OT3)T combined with IL-2
To observe anti-tumor effects in vivo, y982(OT3)T cells were used for experimental immunotherapy in nude mice engrafted with human ovarian cancer. Intratumoral injections of y9δ2(OT3)T cells greatly decreased the growth of tumors, significantly different from PBS control and vector retrovirus particle infected cells control following 18 days'therapy. Intratumoral injections of y9δ2(OT3)T cells prolonged the survival of tumor-bearing nude mice than the other two groups.
In a word, we prepared gene-modified y8T cells using gene engineered technology. In vitro and vivo experiments, gene-modified y8T cells showed the anti-tumor potency, providing a new strategy for immunotherapy.
Depression is the main type of mood disorders, characteristic with a significant and lasting decline in the mood. Depression has high disease incidence and mortality which threatens human health and life. The traditional theory that depression is a consequence of deficient monoamine activity has been revised by the addition of novel theories suggesting that disturbances in other systems are important for the pathophysiology of depression. In particular, there is evidence that glucocorticoids, cytokines and neurotrophins are involved in the manifestation and treatment of this disease. To date, the majority of studies on cytokines in depression have investigated in a single cytokine subset, especially monocytic proinflammatory cytokines such as IL-1β, IL-6 and TNF-a. However, cytokines exert pleiotropic activities and interact closely with each other. Also, individual cytokines belonging to the same subset may have different effects on the immune response. In the present study, we measured 3 subsets of cytokine family, specifically monocytic cytokines (IL-1β,IL-6,TNF-a), Thl cytokines (IFN-y,IL-2) and a Th2 cytokine(IL-4). The depressive patients showed a significant increase in serum levels of IL-1βand IL-6 compared to controls. And the IL-1βand IL-6 production were positively correlated(r=0.308 P<0.01), indicating IL-6 and IL-1βmay have a synergistic effect in the pathogenesis of depression. No significant differences in TNF-a level between both groups were found, although the mean values of this cytokines in MDD patients was slightly increased compared to that in the control subjects. In depressive patients, quantitative measurements were significantly decreased for IFN-y and IL-2 compared to those in controls. In contrast, serum levels of IL-4 were significantly higher in depressive patients, than that in control subjects. In addition, we tested glial cell line-derived neurotrophic factor (GDNF) and MICA expression in the serum of patients with depression. GDNF in patients with depression was significantly lower than that in the control group, and MICA was significantly higher than that of control subjects. Detection of these indicators of depression patients following the six weeks of fluoxetine treatment, there has been a marked restoration change. We believe that the level of GDNF and MICA may be one of the biological indicators. Of course, its etiology, pathological changes and other related mechanisms requires further study.
In conclusion, in the second part of my thesis, we had studied the changes of cytokines, GDNA and MICA in serum of depressive patients, providing clues and basis for the further research on depression.
引文
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