Objective To design and construct the eukaryotic expressed vector of suicide genes, which contained 5 copies of hypoxia-responsive element (5HRE), promoter of alpha-fetoprotein gene (AFPp） and nitroreductase from Escherichia coli. Methods The constructing processes were as follows: ①The design of primer: Suicide genes of NTR in the Escherichia coli, which contained 6his-tag gene (6his-tag), were cloned by overlapping PCR. ②The construction of 5HRE: The single strand of synthetized HRE oligonucleotide was annealed, and 5HRE was constructed by multiple recombinant clone. ③The recombination of NTR gene, 5HRE, AFPp and pIRES2-EGFP: pIRES2-EGFP, which had removed the instant early promoter of cytomegalovirus, was recombined with NTR gene, 5HRE, AFPp. In this way, the eukaryotic expressed vector of pIRES2-EGFP-5HRE-AFPp-NTR, which carried NTR gene, 5HRE, AFPp was finally constructed. Results NTR gene, which contained the fusion of 684-base pair and 6his-tag gene, was cloned successfully, and its sequence was coincident with the result published by Genbank. A 221-base pair of 5HRE was also constructed, which was in accordance with the expected sequences. The integrity of the eukaryotic expressed vector was verified by restriction enzyme digestion and DNA sequence analysis, respectively. Conclusion The eukaryotic expressed vector of pIRES2-EGFP-5HRE-AFPp-NTR is successfully constructed, which may be used for its further investigation in vitro.
Objective To explore the effect and mechanism of sleeve gastrectomy （SG） for type 2 diabetes mellitus （T2DM） in Goto-Kakizaki （GK） rats. Methods Thirteen male GK rats at 12 weeks of age were randomly divided into SG group （n=7） and sham operation group （SO group， n=6）， receiving SG surgery and sham operation respectively.Body weight， food intake in 24hours， fasting plasma glucose， plasma glucagon-like peptide-1 （GLP-1）， and plasma Ghrelin of rats in 2 groups were measured or tested before operation， 1， 4， 10， and 26 weeks after operation. In 10 weeks after operation， fecal energy content of rats in 2 groups was tested， in addition， oral glucose tolerance test （OGTT） and insulin tolerance test （ITT） were performed to investigate the glucose tolerance and insulin sensitivity. Results ①Body weight：there were no significant difference on body weight between the 2 groups （P＞0.05）. Compared with time point of before operation， the body weight of both 2 groups decreased in 1 week after operation （P＜0.01）， but increased in 10 weeks and 26 weeks （P＜0.01）. ②Food intake in 24 hours：compared with SO group， the food intake of SG group were lower in 4 weeks and 10 weeks after operation （P＜0.05）. Compared with time point of before operation， the food intake of SG group were lower in 1， 4， and 10 weeks after operation （P＜0.05）， but lower only in 1 week in SO group （P＜0.05）. ③Value of fasting glucose：compared with SO group， the value of fasting glucose in SG group were lower after operation （P＜0.01）. Compared with time point of before operation， the value of fasting glucose of SG group were lower after operation （P＜0.01）， but decreased in 1 week only in SO group （P＜0.01）. ④Level of serum GLP-1：compared with SO group， the levels of serum GLP-1 in SG group were higher in 4， 10， and 26 weeks after operation （P＜0.05）. Compared with time point of before operation， the levels of serum GLP-1 in SG group were higher in 4， 10， and 26 weeks after operation （P＜0.05）， but levels of serum GLP-1 in SO group didn’t change significantly （P＞0.05）. ⑤Level of serum Ghrelin：compared with SO group， the levels of serum Ghrelin in SG group were lower at alltime points after operation （P＜0.01）. Compared with time point of before operation， the levels of serum Ghrelin in SGgroup were lower at all time points after operation （P＜0.001）， but levels of serum Ghrelin in SO group didn’t change significantly （P＞0.05）. ⑥Areas under curves （AUC）：the AUC of OGTT and ITT test in SG group were both lower than those of SO group （P＜0.01）. Conclusion SG surgery can induce the level of fasting plasma glucose， and canimprove glucose tolerance and insulin sensitivity with significant changes of levels of plasma GLP-1 and Ghrelin， sugg-esting that SG surgery may be a potential strategy to treat patient with T2DM but without obesity or insulin resistance.
Objective To detect expression of Ras association domain family 1A （RASSF1A） gene in the colonic carcinoma tissue and to analyze the relationship of this expression to its clinical features. Methods Immunohistochemistry and Western blot methods were employed for detecting the RASSF1A protein expressions in 34 colonic carcinoma tissues and corresponding normal colon tissues. RT-PCR was employed for detecting RASSF1A mRNA expression. Results ①The RASSF1A protein expression in the colonic carcinoma tissues was significantly lower than that in the normal colontissues by using immunohistochemistry〔35.3% （12/34） versus 97.1% （33/34）， P＜0.05〕.There were significant relati-onships of RASSF1A protein expressions to the tumor differentiation and TNM stage （P＜0.05）， in other words， the positive rates of RASSF1A protein in the moderately and well differentiated andⅠ＋Ⅱof TNM colonic carcinoma tissues were all higher （P＜0.05）. ② The RASSF1A protein expression in the colonic carcinoma tissues was significantly lower than that in the normal colon tissues by using Western blot 〔0.316 8±0.019 6 versus 0.914 4±0.177 6， P＜0.05〕， which was close to the result of RT-PCR〔0.158 9±0.223 7 versus 0.572 3±0.193 9， P＜0.05〕. Conclusions Absentexpre-ssion of RASSF1A gene in the colonic carcinoma tissue might play an important role in tumor genesis and tumor progre-ssion， and it might become useful early detection of the colonic carcinoma.