摘要
本研究首先建立了旋毛虫感染诱导的抗MCF-7乳腺癌细胞的动物模型,采用抑制性消减杂交(SSH)技术成功构建了旋毛虫感染诱导的MCF-7乳腺癌细胞基因表达变化的cDNA消减文库,探索旋毛虫感染诱导的MCF-7乳腺癌细胞基因的表达变化。将BALB/c小鼠分为荷瘤11d前接种旋毛虫组和荷瘤11d后接种旋毛虫组。实验组小鼠分别接种不同剂量的旋毛虫,对照组皮下注射0.2mL生理盐水,每只小鼠的右后肢皮下接种4×106个MCF-7乳腺癌肿瘤细胞,并在荷瘤20d后剖检小鼠,测定小鼠体内肿瘤的体积、重量及抑瘤率,并检测T淋巴细胞亚群。结果发现旋毛虫对BALB/c小鼠体内的MCF-7肿瘤细胞的生长有一定的抑制作用,将两组间抑瘤效果进行比较,经统计学分析,荷瘤11d前接种旋毛虫组的抑瘤效果优于荷瘤11d后接种旋毛虫组,从而建立了旋毛虫感染诱导的抗MCF-7乳腺癌细胞动物模型。以该模型的实验组和对照组的肿瘤组织为研究对象,采用SSH技术成功构建了旋毛虫感染后诱导MCF-7肿瘤细胞上调基因表达消减文库,克隆目的基因片段大小分布在200-500bp之间,20个阳性克隆经测序BLAST比对,获得差异表达的已知功能基因8个和未知功能基因4个,8个为重复检出基因,其中RB基因可能与乳腺癌细胞的生长抑制有密切关系。利用反向Northern杂交技术对所获得基因的特异性表达进行了验证。本研究为深入探讨旋毛虫感染诱导MCF-7乳腺癌细胞基因表达变化与旋毛虫抗肿瘤机理的研究奠定了一定的基础。
Trichinosis is a parasitic zoonoses , it caused by Trichinella spiralis which is a tiny nematode. People infected trichinosis through eating contain trichinella larvae capsule raw or half-Fresh pigs, dogs, wild boar, bear meat or meat products e.g.
Every year, 7million people died of cancer in the world, China has 1 million people lost their lives too. In order to cure the cancer, the scientists made their great efforts. Up to now, we have not find a way to overcome cancer. Cancer, also known as malignant tumors, and relatively are benign. Because of a variety of tumorigenesis factors,local tissue of body dysplasia and form local mass is Tumor. Benign tumors easily cleared away, no metastasis or recurrence, and only have squeeze obstructive role in organs or tissues. But malignant tumors can destruction the structure and function of tissues and organs, induce hemorrhagic necrosis concurrent infection, the patient may death due to organ failure. Breast cancer (mammary carcinoma) from the breast ductal epithelial malignant tumor, the notion that diagnosis and treatment of breast cancer have undergone a great change. Retention breast treatment, sentinel node biopsy, the new adjuvant therapy, molecular targeted therapy, such new technologies and ways enriching the content of breast cancer treatment, the further understand of breast cancer’s Biologic behavior make decision mode of adjuvant therapy changes,final diagnosis method has also diversify. Gene therapy as a novelty treatment for cancer has brought hope. In recent years, many studies of Tumor therapy reported that a variety of parasites and viruses has been shown can inhibit tumor.
There are many reports of domestic data, trichinosis can increase the resistance of host to tumor. Mice can inhibition the tumor cell proliferation in vivo or tumour growth that transplant to vivo after infected Trichinella spiralis .Trichinella spiralis has anti-tumor effect has been confirmed by many experiments, but its anti-tumor molecular mechanism remains unclear.Compared to many subtractive hybridization technique, the advantages of SSH technology mainly in the following aspects:first, Step 1 hat based on the principle of hybridization kinetics make different abundance mRNA molecules achieve convergence, SSH overcome different mRNA molecules on the number of copies to the impact of hybridization results, thereby greatly improving the subtractive sensitivity. Second, the SSH technique not need any intermediate steps dysjunction single- or double-stranded cDNA chain, just one series subtractive hybridizate , can be concentration differentially expressed cDNA molecules to 1,000 times, which simplifies the operation. Third, the false positive rate is more lower. This is the biggest advantage of SSH technology, its Step 2 hybridization and double PCR can guarantee that the cloned have 94% really positive. Fourth, high sensitivity. In the DD and RDA, the low abundance mRNA can not detected in general, SSH by the homogenization method and target fragment of enrichment to ensure the detected of low abundance mRNA. Fifth, high speed, and high efficiency. SSH can be isolated dozens or even hundreds of differentially expressed genes at the same time .
This experiment established Trichinella spiralis infection-induced anti-tumor cells MCF-7 animal model,established an ideal animal model for the selection of differentially expressed genes.
Suppression Subtractive Hybridization (SSH) technology successful constructed Trichinella spiralis infection-induced MCF-7 cell gene expression changes in tumor cDNA library. Inoculated Trichinella spiralis after bearing cancer group as test , the other group as control, extract RNA, reverse transcription and synthesis of single-chain and double-stranded cDNA, the average size of a digestion 400 - 600bp fragment. Driven element group cDNA split into two groups, connected with two different adapter respective, following with driver element cDNA do double subtractive hybridizate and double PCR inhibition amplification, 2nd PCR products after purified connect with pMD-18T vector, and transformed into E.coli DH5αcompetence cell to amplificate library, ultimately constructed with high subtracte efficiency related cDNA library of the Trichinella spiralis anti-MCF-7 cells, gain 60 white positive clones, and randomly selected 30 clones identified by PCR, the results show that the identification of the insert mainly distribute in the 200-500 bp. From the subtracted cDNA library screening of 20 differentially expressed cloning, the sequence that sequencing satisfied with refer to the NCBI (http://www. Ncbi. Nlm. Nih. Gov / VecS Green / VecScreen. html)UniVec database, remove adapter sequences and vector sequence at both ends of sequence, via non-GenBank Redundant DNA database (nr) and the EST database for BLAST comparison, reference relevant literature EST classification criteria, obtained 8 known genes, 5 unknown gene. These genes increased in the expression after infected with Trichinella spiralis, at the same time gained a tumor suppressor gene--Rb gene. RBgene(Retinoblastoma gene)is retinal cell tumor gene for the retinoblastoma susceptibility gene, the first cloned and completion of the entire sequencing tumor suppressor gene in the world. RB gene transcription product about 4.7 kb, the expressed product is protein which composed of 928 amino acid, the molecular weight about 105 kDa, called P105-Rb. Rb protein a kind of DNA-binding protein located in the intranuclear.RB protein phosphorylation / phosphorylation is the main form to regulate cell growth and differentiation. It showed to phosphorylation in the G0, G1 phase, In the G0 ,S, M period in the phosphorylation state.
Rb gene expression increased after infected with Trichinella spiralis, thereby inhibiting tumor growth. Suppressor–tumor gene, also known as anti-cancer gene. Early 1960 s, it was find that the same kind of cancer cells and normal fibroblast cell fusion, acquired the descendants of hybrid cells as long as retain some of the pro-normal chromosomes, can be manifested as normal phenotype, but with the loss of chromosome can re-emergence of malignant cells . This phenomenon shows that the normal chromosome may be contains some tumor suppression gene, and their loss, idiovariation or loss of function made activing oncogenes to produce a marked effect and formed cancer. Suppressor gene product inhibits cell proliferation,promotes cell differentiation and inhibition of migration, playing a negative regulatory role.Usually considered that tumor suppressor gene mutat is recessive.
引文
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