ObjectiveExpression and purification of human tumor necrosis factorα(hTNFα)fusion protein with a stretch of six consecutive histidine residues(6×His) in E. coli.MethodshTNFα fusion proteins with 6×His at N and C terminus were expressed by using E. coli expression vectors pET- 28a(+) and pET-22b(+). The His6 tag allows the expression fusion protein purified in one step by immobilized metal Ni2+ chelation affinity chromatcgraphy in native state. Results The two construct expression vectors were expressed in E. coli respectively, the former with high level as insoluble protein, account for 45% of the total bacteria proteins and not purified; the later 8% as soluble protein, and characterized by SDS- PAGE, Westren-blot. By using affinity chromatography through Ni2+ - IDA Sepharose 6B, 100ml induction culture had 0.4mg hTNFα-6×His fusion proteins. Its purifity reached 90 %. ConclusionThe purification expression product can possess TNF activity and reach 5.42×104U/mg.

Objective To produce bioactive human bone morphogenetic protein-4 by Escherichia coli genetic engineering and investigate the effect of the product, recombinant BMP-4, on the bone marrow stem cells. Methods cDNA of human morphogenetic protein-4 mature peptide was obtained by RT PCR from tissue of human placenta. The gene was constructed in the pET-22b(+) expression vector and expressed in Escherichia coli BL-21 after transformation and induction by IPTG. The harvested protein was proved to be bioactive by inducing ectopic bone information in mouse thigh. The protein was applied to induce the cultured bone marrow stem cell. Shape change of the cell, ability of ALP (alkaline phosphatase) and concentration of OC (osteocalcin) were investigated. Results SDS-PAGE revealed a new protein band that located in position of 14?103 after 4 hours induction, the new protein made 15% of total bacteria protein, the rhBMP-4 could induce the cultured bone marrow stem cell of New Zealand rabbit to differentiate into osteoblasts and form calcified node. The ability of ALP and concentration of OC of tested group increased significantly than that of the control group. Conclusion The bioactive rhBMP-4 can be produced by Escherichia coli genic engineering, this protein can induce bone marrow stem cells to differentiate into osteoblast cells.

AFP from fetal serum and serum of patients with primary hepatic cancer were purified by affinity chromatography. Then, the lentil lectin-reactive and nonreactive variants of these purified glycoproteins were prepared by affinity chromatography with immobilized lectin. Glycopeptides and oligosaccharides were prepared from variants by protease treatment and hydrazinolysis, respectively, and subjected to carbohydrate and amino acid analysis. A small difference in the carbohydrate compositions of each variant was observed. Analysis of chemical compositions of AFP variants is useful for the differential diagnosis between benign liver diseases and primary hepatic cancer.

In order to study the expression and the feasibility of scaled production of neuropeptide in the routine expression system such as E.coli with the pituitary adenylate cyclase activating polypep tide(PACAP) as an example, the following experiments were carried out. First, on the basis of the reported amino acid sequence of PACAP, DNA sequence of PACAP w as deduced and six partially complementary oligonucleotide fragments were design ed. The coding region of PACAP was obtained by renaturing the DNA fragments and ligation and identified by DNA sequencing. The coding region of PACAP was cloned into plasmid pGEX-4T-3 and transformed into E.coli BL21(DE3 ). An expression strain BLPACAP was selected. SDS-PAGE analysis revealed that t he GST-PACAP fusion protein was highly expressed and accumulated to about 30% o f the total bacterial proteins. By affinity chromatography, up to 90% GST-PACAP was purified by one step from bacterial lysate. The purified protein could prom ote neurite outgrowth of PC12 cells and the survival of spinal cord neurons.

Objective To evaluate total gastrectomy and regular lymph node dissection for the treatment of advanced cardiac cancer. Methods Three hundred and eighty-seven patients with advanced cardiac cancer underwent total gastrectomy D2+ procedure. The relationships between depth of invasion, lymph node metastasis,the number of metastastic lymph nodes dissected and postoperative survival rate were analyzed. Results The 3-year and 5-year survival rates were 47.3% and 34.2% , respectively. Metastatic rate of N3 lymph node was 4. 8% and 15. 2% in advanced cardiac patients of PT3 and PT4, respectively. Survival rates significantly decreased along with the increase of lymph node metastasis. The 3-year and 5-year survival of patients who had less than 15 lymph nodes dissected was significantly poorer than those who had more than 15 lymph nodes dissected, respectively. The postoperative complication rate and mortality rate of the two groups were 14. 2% and 2. 52% , respectively. Conclution To improve the quality of life and survival rate, D2 + radical total gastrectomy should be performed for advanced cardiac cancer, and when indicated resection of spleen and/or body and tail of the pancreas should be performed.

Objective To explore the predictive value of microvessel density(MVD)and blood vessel invasion(BVI)in hepatic metastasis from early-stage rectal cancer.Methods MVD and BVI in the tumor tissue from 380 patients with stage I and II rectal cancer was determined by immunohistochemical S-P method with anti-CDIOS antibody and anti-CD34 antibody,respectively.Multinomial logistic regression was performed to analyze the predictive value of MVD and BVI in hepatic metastasis from early-stage rectal cancer.Results CD105 was expressed in newborn blood vessels,not in normal blood veseels.in the rectal cancer tissue.MVD was correlated with histological type and infiltration depth(P＜0.05).Besides histological type and infiltration depth,BVI was also correlated with histological grade.Multivariate analysis revealed that histological type,tumor infiltration depth,BVI,adjuvant therapy,and MDV were independent predictors of hepatic metastasis from rectal cancer.The risk of hepatic metastasis in patients with postive expression of either MVD or BVI or both were significant higher than that in patients with low expression of MVD and those without BVI expression[hazard ratio(95%CI),4.210(2.182-11.214)].Conclusion BVI and MVD are independent predictors of hepatic metastasis from stage I and II rectal cancer.Combined detection of MVD and BVI may help to predict the clinical outcome of patients with early-stage rectal cancer.

Objective To detect the clinical factors related with liver metastasis in young patients with rectal cancer.Methods Three hundred and fifty young patients with rectal cancer were collected to set up the database.Single and multi-factor Logistic regression was applied to indicate the independent factors relating to liver metastasis.The regression equation to predict probability of liver metastasis from rectal cancer was established.Results Liver metastasis was 120 cases (34.3%).Single-factor analysis revealed that patho-organization type,pathologytype,infiltration extent,blood vessel invasion (BVI),TNM stage,operation character,the preoperative level of carcino-embryonic antigen,histology grading were related with liver metastasis.Multi-factor analysis revealed that only BVI (P=0.001),TNM stage (P=0.001),pathoorganization type (P=0.005),the preoperative level of CEA (P=0.008) and operation character (P=0.032) were independent factors to predict probability of liver metastasis.Conclusions Rectal cancer of young patients who being with BVI,advanced phase,high preoperative level of CEA,radical operation or poor differentiation degree,are apt to develop liver metastasis.They should be given further individualized intensive adjuvant treatment.

OBJECTIVETo study the result of surgical treatment of benign ulcer of the gastric cardia.

METHODSClinical symptom, number, position and size of ulcer, preoperative X-ray gastroscopic examine, type and complication of operation were retrospective analyzed in 21 patients with benign ulcer of the gastric cardia.

RESULTSAverage age in 21 patients was 60. Ulcer complicated bleeding in 7 cases (33.3%) and perforation in 5 cases (23.8%). Diagnostic rate of preoperative X-ray and gastroscopic examine was 57.1% and 100%, respectively. Of 21 patients 18 (85.7%) were treated by upper partial gastrectomy, and 3 (14.3%) by total gastrectomy. All were not operative death and anastomotic leak.

CONCLUSIONSupper partial gastrectomy can resect focus of ulcer and decrease secretion of stomach acid. It is a simple and ideal pattern of operation for patients with benign ulcer of the gastric cardia.

Adult ; Aged ; Cardia ; Female ; Humans ; Male ; Middle Aged ; Prognosis ; Stomach Ulcer ; surgery