【作者】 单延红；

【导师】 崔满华；

【作者基本信息】 吉林大学 ， 妇产科学， 2011， 博士

【摘要】 卵巢癌是妇女第五大癌症死亡原因,是妇科癌症首要死亡原因,一生当中大约每58个妇女就有一个患卵巢癌。由于卵巢位于人体内部,卵巢癌早期无明显症状,发现时多已为晚期。主要采用手术切除及顺铂联合紫杉醇进行化疗。细胞减灭术提高晚期患者生存率的作用有限,而化疗副作用很大,对于Ⅲ、Ⅳ期的病人在一线治疗后平均存活期大约为18个月。一些晚期患者由于身体素质差,不能耐受化疗。因此,临床上需要更好的辅助治疗方法,延缓复发和提高卵巢癌患者的生存率。过继免疫治疗作为肿瘤生物治疗的一种模式,通过输注免疫活性细胞以增强肿瘤患者的免疫功能,为晚期卵巢癌患者,尤其是复发的卵巢癌患者及身体素质差,不能耐受化疗的患者提供了新的治疗途径。人类细胞因子诱导的杀伤细胞(cytokine-induced killer,CIK),是体外由不同的细胞因子如CD3单克隆抗体,IL-2,IL-1和γ-干扰素等诱导的免疫效应细胞,大量实验研究及临床试验表明,CIK细胞作为一种过继免疫治疗因子能够有效抑制和杀伤肿瘤细胞,与手术或放、化疗结合,可以防止癌的扩散和复发。但CIK细胞与中药相结合抗肿瘤的研究,国内外鲜有报道。姜黄素(Curcumin,Cur)是从姜科植物姜黄的根茎中提取的有效成分之一,有广泛的药用价值,抗癌是其主要的作用之一。本研究通过观察CIK细胞联合Cur对卵巢癌SKOV3细胞的增殖抑制作用及对细胞凋亡的影响,探讨二者联和使用是否具有协同抗肿瘤作用,从而强化CIK细胞作为过继免疫治疗的优势,并探讨其可能的作用机制,为卵巢癌细胞的免疫治疗及中药治疗提供实验研究,为卵巢癌的综合治疗提供一个新的治疗思路,为进一步临床应用奠定基础。方法：首先诱导脐血CIK细胞,将SKOV3细胞随机分为Cur组、CIK细胞组、Cur联合CIK细胞组,MTT法测定各组细胞的增殖抑制率,并进行联合应用效应计算；电镜下观察各组细胞凋亡形态,流式细胞仪检测细胞凋亡,RT-PCR检测各个给药组对SKOV3细胞FAS,FADD,CASP3mRNA表达的影响,Weston blot检测Cur作用后卵巢癌细胞表面Fas表达及CIK表面FasL的表达,MTT法检测抗FasL单克隆抗体对CIK细胞及Cur联合CIK细胞对卵巢癌细胞增殖抑制率的影响。结果：(1)实验中选择较低浓度的CIK与Cur,其单独作用于SKOV3细胞,抑制率均较低,而二者联合应用后能明显促进对增殖的抑制,且随着药物浓度及效耙比增加及作用时间的延长,抑制率也逐渐增加,即呈时间和浓度依赖关系。在效靶比为12.5：1,Cur浓度为20μmol·L-1时,作用48小时,单独应用的抑制率分别为48.7%及41.9%,而二者联合应用后抑制率达到76.2%,为单独应用的1.6倍及1.8倍。且联合应用效应Q值显示：Cur与CIK细胞合用作用于SKOV3细胞后Q值均大于或等于1.2,进一步证明二者合用具有协同抗肿瘤效应。(2)透射电镜显示：对照组卵巢癌细胞大小不等,细胞表面可见少量微绒毛,可见细胞分裂相和多核巨细胞。而各个给药组作用24h后,均呈现典型的细胞凋亡的形态学改变,Cur联合CIK组细胞凋亡的形态学改变最明显,Cur组细胞大小不等,胞质内多见电子致密物,部分细胞线粒体和粗面内质网成泡状改变,胞质内可见空泡结构,核内特染色质增多,成块状；CIK组细胞大小不等,细胞膜微绒毛减少,少量细胞膜有破损,胞质内可见较多的空泡结构和电子致密物,线粒体空泡样改变；Cur联合CIK组细胞大小不等,细胞数明显减少,细胞膜有破损,少见多型核巨细胞,胞质内可见大量的空泡结构或电子致密物,线粒体脊断裂或呈空泡样改变,细胞核固缩。表明Cur与CIK联用具有明显诱导SKOV3细胞凋亡的作用。(3) RT-PCR检测CIK细胞、Cur、及Cur联合CIK对SKOV3细胞内Fas的mRNA表达影响。结果显示,与对照组相比,DMSO和Cur组对Fas的mRNA表达无影响,而CIK细胞组和Cur联合CIK组能够促进FAS的mRNA表达(P<0.05),表达阳性率分别为对照组的120%及186%；Cur联合CIK组FAS的mRNA表达明显增加,为CIK细胞组的1.55(186/120)倍；与对照组相比,各个给药组对FADD的mRNA表达无影响；与对照组相比,DMSO对Caspase3的mRNA表达无影响,而Cur组、CIK细胞组和Cur联合CIK组均能够促进Caspase3的mRNA表达(P<0.05),表达阳性率分别为对照组的115%、205%、302%,且Cur联合CIK组Caspase3的mRNA表达明显增加,分别为Cur组及CIK细胞组的2.62(302/115)倍及1.47(302/205)倍。(4) Weston blot检测Cur作用后卵巢癌细胞表面Fas表达。结果显示,与对照组相比,DMSO对Fas蛋白表达无影响,而5、10和20μmol·L-1 Cur能够促进SKOV3细胞Fas蛋白表达(P<0.05)。表达阳性率分别为对照组的168%,253%及249%。(5) Weston blot检测Cur对CIK细胞膜FasL蛋白表达的影响。结果显示,与对照组相比,DMSO对FasL蛋白表达无影响,而5、10和20μmol·L-1 Cur能够促进CIK细胞膜FasL蛋白表达(P<0.05),表达阳性率分别为对照组的139%,142%及138%。(6)MTT检测抗FasL单克隆抗体对卵巢癌细胞增殖抑制率的影响。通过FasL抗体预先孵育,阻断了Fas/FasL途径,CIK组和Cur联合CIK组FasL抗体预先孵育能够明显抑制对SKOV3细胞的增殖抑制作用。抑制率分别由31.4%降至21.1%,69.8%降至51.6%。结论：(1)脐血来源的CIK细胞体外增殖快,单个核细胞提取率高,对卵巢癌SKOV3细胞杀伤活性强。可方便地用于临床治疗。(2)Cur与CIK细胞联合使用,作用于卵巢癌SKOV3细胞,体外具有协同抗肿瘤效应。作为一种辅助治疗手段,为肿瘤患者,尤其是复发的及不能耐受化疗的晚期肿瘤患者带来了希望。(3)Cur与CIK细胞联合使用,诱导SKOV3细胞凋亡作用增强,表明二者合用具有较好的肿瘤治疗效果,有希望成为发展前景良好的综合治疗方法。(4)Cur与CIK细胞联合使用,诱导卵巢癌细胞凋亡的机制可能为Cur促进SKOV3细胞表面Fas表达增强,CIK细胞表面FasL表达增强,从而使SKOV3细胞内Fas表达增加,最终使caspase3表达增高。为二者的进一步临床应用提供了理论依据。本研究将Cur与CIK细胞联合使用,作用于卵巢癌SKOV3细胞,增强了对SKOV3细胞的增殖抑制作用,强化了CIK细胞作为过继免疫治疗的优势,为晚期卵巢癌患者,尤其是复发的卵巢癌患者及身体素质差,不能耐受化疗的患者提供了新的治疗途径。为卵巢癌的综合治疗提供一个新的治疗思路：在卵巢癌治疗的具体方案设计上可采取多种方法联合应用综合治疗。更多还原

【Abstract】 Ovarian cancer occurs with all lifetime incidence in approximately 1 in 58 women and it is the fifth leading cause of cancer death in women and is the leading cause of death among gynecologic cancers. Ovarian cancer has no obvious syndrome and most patients are diagonosed at late stages due to ovary lies in inner part of the body. Surgery and chemotherapy of Cisplatin(CDDP) combined with paclitaxol are mainly be used. The effect of survival rate upon patients at late stages be improved with cytoreductive surgery are limited, and chemotherapy has much side-effect, some patients at late stages can not tolerant chemotherapy due to poor physical. So it is urgently needed to find a more efficient adjunctive therpy to delay recurrenc and enhance survival rate of ovarian cancer patients.Cytokine-induced killer(CIK) cells are immune effector cells which are induced by various cytokines such as monoclonal antibody(mAb), interleukin-2 (IL-2), IL-1, and interferon-gamma (IFN-y) in vitro. A large number of experiment research indicated that CIK cells can effectively restrain and kill tumor cells, and can prevent tumor from spreading and recurance when combined with surgery, radiotherapy and chemotherapy. But the study on antitumor of CIK cells combined with traditional Chinese medicine is rarely reported at home and abroad.Curcumin (Cur) is one of effective ingredient which is abstracted from the curcuma rhizome of curcuma genus in ginger family, which has broad-spectrum pharmaceutic value, and anti-tumor is one of the major effect.Adoptive immunity therapy act as one kind of biotherapy mode on tumor, by means of transfusing immunocompetent cell to enhance tumor patients immunologic function, provide a new treatment way for late stage ovarian carcinoma patients,especially recurrent ovarian carcinoma patients and those who has poor physical fitness failure to tolerance chemotherapy.This study was designed to observe inhibitory effects on the proliferation of Curcumin associated with CIK cells on ovarian carcinoma SKOV3 cells in vitro and to investigate wheather they have synergetic anti-tumor effects if they are used together thereby strengthen CIK cells superiority on adoptive immunity therapy and to discuss the possible mechanism, so that provide experimental study for traditional Chinese medicine and immunotherapy on ovarian cancer, provide a new remedy thingking for comprehensive treatment on ovarian cancer, to lay the foundation for more clinical pratice.Methods:CIK cells were induced from umbilicus cord blood firstly, and the SKOV3 cells were randomly divided into Cur group, CIK group, Cur associated with CIK group. The inbition ratio on the proliferation of each group was checked with MTT methods, and the effect of joint use was calculated. The apoptosis morphology of each group cells was observed under electron microscope. Flowcytometric analysis was used to dectect cell apoptosis. The mRNA level of Fas, FADD, Caspase3 of each group of drug administration was measured by RT-PCR. The Fas level on ovarian cells surface and the FasL level on CIK cells surface after Curcumin function on them were detected with Weston blot. The level of inbition ratio on proliferation of CIK cells and CIK associated with Cur after FasL monoclonal antibody being administrated was checked with MTT methods.Experimental result:(1) The lower concentration of CIK and Cur were choosed in the study, the inhibition ratio of each group was lower when they functioned on SKOV3 cells alone, but the inhibitory effect on proliferation was promoted obviously when they were used together, and inhibition ratio was also gradually enhanced with the rise of medicine concentration and the ratio of effector contrast to target and time, that is they present time and dose depence relation. The inhibition ratio of SKOV3 cells was respectively 48.7% and 41.9% when they act alone for 48 hours by the concentration of CIK of effector to target ratio of 12.5:1 and Cur at 20μmol·L-1, but it approached 76.2% when they were used together, was 1.6 times and 1.8 times compared to that when been applied alone.And the value of Q of joint use effect reveal that the value of Q all greater than or equal to 1.2 after Cur and CIK were used together on SKOV3 cells, more to move forward to prove they have synergetic anti-tumor effect when they were used together.(2) Transmission electron microscope show that the ovarian cancer cell size was unequal in the control group,a little of microvilli was visible on the cell surface,cell division photo and multinuclear giant cells were visible, but each group been drug administrated for 24h without exception appeared a change on apoptosis morphology, the change of apoptosis morphology in the group of Cur associated with CIK was most obvious, the cell size was unequal in the group of Cur, electron densification matter was mostly seen, part of cell mitochondria and asperities endoplasmic reticulum became vacuole shape, vacuole structure was visible in the cytoplasm, exceptional chromatin rise in the nucleus, in bulk shape; the cell size was unequal in the group of CIK, cell membrane microvilli reduced,a small amount of cell membrane damaged, much vacuole structure and electron densification were visible in the cytoplasm, mitochondria became vacuole shape; the cell size was unequal in the group of Cur associated with CIK, cell count obviously decreased, cell membrane damaged, polymorphism nucleus giant cell was scarce, a mass of vacuole structure or electron densification were visible in the cytoplasm,mitochondria ridge ruptured or became vacuole shape, nucleus pyknosis.It make clear that Cur combined with CIK can induce SKOV3 cells apoptosis obviously.(3) The mRNA level of Fas, FADD, Caspase3 of SKOV3 cells after CIK, Cur, CIK associated with Cur act on SKOV3 cells was measured by RT-PCR. The result show that the mRNA level on Fas of DMSO and Cur group had no obvious change, the mRNA level on Fas was promoted in the group of CIK and CIK associated with Cur compared with control group (P<0.05), the positive rate was respectively 120% and 186% upon control group. the mRNA level on fas was increased sharply in the group of Cur associated with CIK, was 1.55(186/120) times upon CIK cells group. The mRNA level of FADD in each group of drug administration had no obvious change compared to control group. Compared to control group, the mRNA level of Caspase3 of DMSO group had no obvious change, but the group of Cur, CIK and Cur associated with CIK could promote the mRNA level of Caspase3 (P<0.05), the positive rate was respectively 115%,205%,302% upon the control group, and the mRNA level of the group in Cur associated with CIK obviously increased, was respectively 2.62 times(302/115) upon the group of Cur and 1.47 times (302/205) upon the group of CIK.(4) The Fas level on ovarian cells surface after Curcumin function on them was detected with Weston blot.The result show that the level of Fas protein in the group of DMSO had no obvious change compared with the control group, but Cur of 5,10 and 20μmol·L-1 could promote the level of Fas of SKOV3 cells (P<0.05). The positive rate was respectively 168%,253%, and 249% upon the control group.(5) the FasL level on CIK cells surface after Curcumin function on them was detected with Weston blot. The result show that the level of FasL protein in the group of DMSO had no obvious change compared with the control group, but Cur of 5,10 and 20μmol·L-1 could promote the level of FasL protein of cell membrance of CIK cells (P<0.05), the positive rate was respectively 139%, 142% and 138%.(6) The level of inhibition ratio on proliferation of CIK cells and CIK associated with Cur after addition of anti-FasmAb was checked with MTT methods. The pathway of Fas/FasL was blocked after addition of anti-FasmAb, The group of CIK and Cur associated with CIK could be restrained obviously the inhibitory effect on proliferation on SKOV3 cells after addition of anti-FasmAb. The inhibition ratio respectively drop from 31.4% to 21.1%, 69.8% to 51.6%.Conclusion:(1) CIK cells extracted from umbilicus cord blood proliferate fast in vitro, extraction ratio of mononuclear cells is high, the killing activity on ovarian cancer SKOV3 cells is powerful. It can be expediently used to clinic treatment.(2) Cur associated CIK has synergic effect of anti-tumor on SKOV3 cells when they are used together in vitro. It bring about hope for tumour sufferer, especially later period tumour patients who is recurrent and failure to tolerate chemotherapy as an adjuvant therapy means.(3) The effects of inducing apoptosis of SKOV3 cells are increased when Cur and CIK are used together, it make clear that they have preferable therapeutic effect when be used together, and it is prominsing to become favourable prospect comprehensive trerapies.(4) The mechanism of inducing apoptosis of Cur and CIK maybe that Cur promote the expression of Fas on SKOV3 cells surface and FasL on CIK cells surface, so that raise the expression of Fas in SKOV3 cells, finally the expression of Caspase3 rise. It offers theory proof for they further clinical practice.The study strengthen the superiority of CIK cells as an adoptive immunity therapeutic by make use of Cur and CIK cells together on ovarian carcinoma SKOV3 cells, which enhance the inhibitory effect on SKOV3 cells. It can provide a new remedy thinking for comprehensive treatment on ovarian cancer: the comprehensive treatment of various means being used jointly should be adopted on specific project design of ovarian cancer therapy.更多还原