摘要
目的：在肿瘤的生长和转移中，淋巴细胞在肿瘤部位的浸润是宿主抗肿瘤免疫应答的重要表现。这种现象被认为与机体抗肿瘤免疫有关，提示人们设想如果诱导更多淋巴细胞浸润到肿瘤局部，是否能起到加强机体抗肿瘤免疫的作用。
Fractalkine (FKN), 又称为“神经活化素”(neuroactin)，作为一种特殊的化学趋化因子，具有膜结合型和游离型两种形式，各自发挥着促使外周血白细胞游走及再循环，并能直接介导白细胞的粘附作用过程而无需粘附因子的参与。FKN作为一种CX3C趋化因子家族中的唯一一员，具有强烈的趋化T淋巴细胞、单核细胞及NK细胞功能，在机体的炎性反应和免疫应答过程中发挥着重要作用。本研究通过克隆并构建FKN真核表达载体，转染神经母细胞瘤细胞系NXS2，从而建立相应的神经母细胞瘤荷瘤鼠动物模型。通过FKN转基因修饰肿瘤细胞的方式，观察其诱导的抗肿瘤效应及其免疫应答。旨在研究FKN体内诱导机体产生抗肿瘤免疫应答的效应及机理，并为进一步探索FKN及其嵌合蛋白作为一种全新的抗肿瘤及抗病毒感染治疗性疫苗提供实验基础和依据。
方法：
1、 FKN基因克隆及鉴定：从上皮来源的小鼠乳腺癌细胞D2F2提取总RNA，通过RT－PCR方法，扩增出FKN cDNA基因片段，经T－A克隆，直接将该片段克隆入T－A克隆载体pCR2.1TOPO，构建重组质粒载体pCR2.1-FKN，在此基础上进行限制性内切酶分析，并通过DNA序列测定分析碱基序列。
2、 FKN真核表达载体构建及其在神经母细胞瘤细胞系NXS2细胞中的表达：选择真核表达载体pIRES，从pCR2.1TOPO-FKN上将FKN cDNA基因片段定向亚克隆至pIRES，通过限制性内切酶分析确证FKN克隆正确，从而成功构建FKN真核表达载体pIRES-FKN。采用阳离子脂质体转染方法，转染神经母细胞瘤细胞系NXS2，用G418加压并以有限稀释法筛选出高效表达FKN的抗性细胞克隆。用RT－PCR检测FKN在NXS2细胞中的转录；ELISA及间接荧光流式细胞法（FACS）分别检测FKN蛋白的分泌型和膜结合型。
3、 转染FKN的神经母细胞瘤动物模型建立及FKN抗瘤作用机制的研究：选择A/J，H-2KK，6－8周龄小鼠，利用神经母细胞瘤致瘤特性，建立三组实验动物模型，即：①NXS2 (原发型)，②NXS2－mock (空载体)，③NXS2－pIRES－FKN。分别观察表达及不表达FKN的肿瘤生长的生长特性。手术完整地摘除肿瘤后，通过RT-PCR，免疫组织化学检测FKN在各组肿瘤中的转录，表达。用免疫组织化学检测各组肿瘤中淋巴细胞的浸润情况。术后第三周，处死小鼠后，肉眼和光镜下观察各组中神经母细胞瘤在肝脏中的转移。
结果：
1 FKN基因克隆及鉴定：从上皮来源的小鼠乳腺癌细胞D2F2提取总RNA，通过琼脂糖凝胶电泳及紫外分光光度法鉴定可获高纯质RNA，以RT－PCR法可扩增出约1200bp大小的片段，与大小为1188bp的FKN cDNA基因大小一致。运用T－A克隆技术，将该片段克隆至pCR2.1TOPO载体，经限制性内切酶分析证明该片段大小与FKN cDNA大小一致，并通过DNA序列测定分析进一步证明该片段硷基序列与gene bank 中公布的小鼠FKN cDNA序列相符。
2 FKN真核表达载体构建及其在NXS2细胞系中的表达：通过定向克隆的方法，从pCR2.1-FKN上将FKN cDNA基因片段亚克隆至pIRES，经酶切鉴定证实FKN cDNA基因片段大小和内切酶位点均正确，FKN真核表达载体pIRES-FKN构建成功。运用脂质体转染方法，转染NXS2细胞系，经G418加压培养和两次有限稀释法筛选出稳定且高效表达FKN的细胞克隆。经PT－RCR法鉴定该细胞内有FKN转录；ELISA和间接免疫荧光流式细胞法（FACS）可分别检测到分泌型与膜结合型的FKN蛋白。
3转染FKN的神经母细胞瘤动物模型建立及FKN抗瘤作用机制的研究：在NXS2的同基因型 A/J小鼠上分别成功建立了三组实验动物荷瘤鼠模型：①NXS2(原发型)，②NXS2 -mock(空载体转染细胞)，③NXS2-pIRES-FKN(表达FKN转染细胞系)。绘制肿瘤生长曲线，可发现FKN表达组肿瘤生长受到明显抑制，该组肿瘤湿重低于其它两个对照组。用RT－PCR和免疫组织化学方法可检测出FKN在肿瘤组
织中的转录及表达。免疫组织化学方法也可以检测到在表达FKN组的肿瘤中有大量的CD4，CD8， CD45阳性细胞浸润。肉眼和光镜下都可以观察到神经母细胞在表达FKN组的小鼠肝脏中无或仅有轻度转移。
结论：
1、 通过基因克隆方法可以直接从上皮来源的小鼠乳腺癌细胞D2F2中克隆出小鼠FKN的cDNA基因。
2、 选择真核基因表达载体如pIRES，通过定向克隆的方法，可成功构建FKN的真核表达载体，pIRES-FKN。
3、 运用真核细胞转染法，转染真核细胞，即乳哺动物细胞，可获较高表达FKN的稳定细胞克隆。在本实验中选择神经母细胞瘤细胞系NXS2，获得FKN的稳定表达细胞系NXS2－FKN。这一FKN高表达细胞系，为肿瘤模型的建立及FKN抗肿瘤免疫应答研究提供了实验基础。
4、 在NXS2同基因型小鼠，A/J小鼠上，运用原发型NXS2，高效表达的NXS2转染细胞系，可成功建立表达及不表达FKN的荷瘤鼠动物模型。
5、 通过观察神经母细胞生长及其对肝脏的转移情况，FKN在实验动物荷瘤鼠模型中，显示出有明显抑制肿瘤生长及转移的生物学效应。
FKN抗肿瘤生长作用可能是通过其特?
Objective: Tumor infiltrating leukocytes are involved in development and growth of tumors and their metastasizes. The origin of these leukocytes may be associated with residual evidence of the host's ineffective immune response to reject the tumor; thus, enhancing the amount of infiltrating leukocytes might lead to an improved host-antitumor effect.
Fractalkine, which is also called neuroactin, is the sole member of the CX3C chemokine subfamily, known to induce both adhesion and migration of leukocytes. The membrane-bound version of fractalkine rapidly induces firm adhesion of CX3CR1-expressing cells under static and flow conditions without requiring selectin-mediated rolling or activation of integrins. In addition, soluble fractalkine released from the cell surface by proteolytic cleavage induces migration of CX3CR1-expressing cells in a similar way to other soluble chemokines.
In this study, mouse FKN was cloned and NXS2 neuroblastoma cells was genetically engineered to express FKN by transfection with FKN mammalian expression vector. The antitumor effect of FKN was monitored in tumor-bearing mouse model which was established by NXS2-FKN in A/J mice. With these data, we tried to test the hypothesis that fractalkine secreted by tumor cells may influence trafficking of leukocytes and subsequently, tumor growth and may open possibilities for designing novel interventions in antitumor and antivirus gene therapy.
Method:
1.Clone and identification of mouse FKN: The coding sequence of mouse FKN was amplified from total RNA which was extracted from D2F2, a mouse breast cancer cell line, by RT-PCR and then cloned into PCR2.1-TOPO vector through A-T clone. The size and the sequence of this insert were verified by restriction enzyme digestion and molecular sequencing analysis.
2.Construction of FKN mammalian expression vector and expression of FKN in tumor cells: To express FKN protein, FKN cDNA was subcloned from pCR2.1TOPO-FKN to a mammalian expression vector pIRES (pIRES-FKN). The length and location of FKN cDNA in this vector was verified by restriction enzyme digestion. Expression of FKN was selected in the presence of G418 selection media and two successive rounds of serial dilution and expansion after pIRES-FKN was transfected into NXS2 neuroblatoma cells with superfect. Transcription of FKN in three different NXS2 cell lines (NXS2-wild type, NXS2-mock, NXS2-FKN) was detected by RT-PCR. The soluble and the membrane-bound forms of FKN were
demonstrated by ELISA and flow cytometry, respectively.
3.Analysis of anti-neuroblastoma function of FKN in tumor-bearing mouse model:On syngeneic female A/J mice, primary tumors were induced by subcutaneous injection of three kinds of NXS2 cells (NXS2-wild type, NXS2-mock, NXS2-FKN) at the dosage of 2×106 in the left flank and tumor growth was monitored by microcaliper measurement. Tumors were removed on the 14th days after their inoculation. Tumors were weighed and transcription as well as expression of FKN in these three groups of tumors were detected by RT-PCR and immunohistochemistry. To characterize the recruiting lymphocytes at the tumor sites, cryopreserved tumor samples were stained by anti-CD4, anti-CD8 and anti-CD45 through immunohistochemistry. 3 weeks after surgery, livers were harvested and spontaneous liver metastatic colonies were counted. For histologic evaluation, 5um sections from paraffin-embedded liver tissues were stained with hematoxylin and eosin.
Results:
1.Clone and identification of mouse FKN: Total RNA was extracted by Rneasy Minikit (Qiagen) from D2F2 cells. The high concentration and purity were determined by measuring the absorbance at 260 nm and 280 nm (A260/A280) in a spectrophotometer. On an agarose gel, DNA contamination is not visible; meanwhile the integrity and size distribution were qualified. By RT-PCR from this tRNA, a fragment around 1200bp long was got which was then cloned into pCR2.1TOPO vector by A-T clone technique. Restriction enzyme digestion by MluⅠand NheⅠ verified the right size of the insert.
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