0 引言 脑脂肪酸结合蛋白(BLBP/B-FABP,FABP7)通常在处于发育期中枢神经系统中的放射状胶质细胞和已分化的成年胶质细胞群例如神经胶质介膜细胞和Bergmann胶质细胞中表达[1-2],在成人中枢神经系统中的表达明显减少[3].有研究发现FABP7的表达对构建和维持放射状神经胶质纤维系统是必需的[4].乳腺癌细胞和脑细胞同样富含脂肪,我们通过观察FABP7基因对人乳腺癌MCF-7细胞增殖能力的影响,为进一步研究FABP7的功能提供依据.

Background: At present most chips use the glass film base by Cy3 and Cy5 mark, their price is so high that are only applied in scientific researches, and clinical application cannot be promoted massively. The topic-based group uses the nylon membrane as the carrier to manufacture gene chips, in order to obtain one kind of chip that has high sensitivity and accuracy, fast simple, may simultaneously examine many kinds of illnesses to get sick, and costs low. Objective: To investigate the feasibility of taking the nylon membrane as the carrier to obtain gene chip, and simultaneously optimize the quantity of the probe and the goal gene. Design, time and setting: Experiments at genetic engineering probe design were carried out from October 2005 to September 2006 in the laboratory of Lanzhou University Science and Technology Garden Baiyuan Gene Company. Materials: Fresh colon cancer samples were excised from Gansu Cancer Hospital between October 2005 and April 2006, while the normal tissues apart from tumor edge above 5-10 cm were taken as controls. The pathology confirmed that the cancer cell had not infiltrated. The nylon membrane belt (from Amersco Corporation) was positive charge. Methods: 200 mg cryopreserved tissues were taken out of liquid nitrogen, total RNA extraction was performed using Trizol method, and mRNA was purified through the Oligo dT chromatography. The first and the second chain of cDNA was synthesized with reverses transcription by using TaKaRa's M-MLV Rtase cDNA synthesis Kit (code D6130). The product was divided into two parts, a part as the template for the digoxin mark PCR, the obtained product served as the goal gene. Another part was taken as the template to manufacture probe. As for the resultant GAPDH, Actin, CyclinD1, the gene fragment PCR product was processed into the purification. Using the positively charged nylon membrane as the carrier, three kind of gene fragment at different density were taken as probes on the carrier, the obtained chip hybridizated separately with the different densities of digoxin mark goal gene. Results: When the goal gene (2.5 μ L and 1.25 μ L) was sampled and added with the hybrid fluid, the goal gene's density was so small as to increase the density on each probe, which was unable to reflect the correct colored tendency. When the goal gene was sampled 5 μ L and the probe density was 10-20 μ L, three kinds of probes exhibited a colored depth tendency arranging from strong to weak, ACTIN>CyclinD1>GAPDH. When the goal gene was sampled 10 μ L, the probe density was 15 μ L, 20 μ L was a reasonable dose to reflect a strong-weak tendency. When the gord gene was sampled 20 μ L, the density of goal gene in the hybrid fluid was excessively high regarding ACTIN and CyclinD1, which was unable to reflect the changes of coloration depth. Conclusion: Using the nylon membrane as the carrier to produce gene chip, the chip background is clear. The reasonable amount of goal gene used is 5 μ L, with the optimized probe amount which it matches is 10-20 μ L.

AIM: To construct eukaryotic expression vector against human cartilage glycoprotein-39(HCGP-39,YKL-40)and to study its effect on proliferation of gastric cancer cell line SGC-7901.METHODS: YKL-40 was obtained from breast cancer tissue by RT-PCR method.The digested double strands DNA were inserted into the downstream of promoter of linearized pcDNA3.1.After it was confirmed by restrictive enzyme digestion method and DNA sequence analysis,the recombinant plasmid was transfected into SGC-7901,a gastric cancer cell line,by using calcium phosphate.The cell proliferation was determined by MTT method.The level of YKL-40 mRNA in SGC-7901 cells was detected by RT-PCR assay.Cell cycles were assayed by FCM analysis.RESULTS: The expression vector of YKL-40 was constructed successfully.The recombinant plasmid enhanced the proliferation of SGC-7901 cells dramatically.The result of RT-PCR showed that YKL-40 mRNA level was increased evidently.The percentage of cells in S phase increased.CONCLUSION: The eukaryotic expression vector of YKL-40 is successfully constructed and it can enhance the proliferation of SGC-7901 cells.

目的:构建针对人类软骨糖蛋白-39(HCGP-39,YKL-40)的真核表达载体,观察其对肿瘤细胞增殖的影响.方法: 采用逆转录方法从乳腺癌组织中获得基因YKL-40,双酶切后插入线性化的pcDNA3.1载体真核启动子下游,重组体经限制性内切酶及测序鉴定,将阳性克隆的载体转染胃癌细胞株SGC-7901,采用噻唑蓝(MTT)法检测细胞的增殖活性、细胞计数并观察细胞生长情况、半定量RT-PCR检测基因YKL-40 mRNA的表达、流式细胞术检测SGC-7901细胞的周期变化情况.结果: 限制性内切酶及测序鉴定表明成功地构建了针对基因YKL-40的真核表达载体;将其转染胃癌细胞SGC-7901后,发现细胞增殖加快,差异具有统计学意义(P<0.05);RT-PCR检测基因YKL-40 mRNA的表达增加;流式细胞检测S期细胞增多.结论: 成功地构建了针对基因YKL-40的真核表达载体,该载体可使胃癌细胞株SGC-7901增殖加快.