Objective To assess functional affinity of humanized anti-HBsAg Fab in order to provide theoretical basis for the radioimmunotherapy of liver cancer bearing HBV. Methods With cognizance of the advantages of solid phase method in time consumption and convenience, we assessed the functional affinity of engineered anti-HBsAg Fab with non-competitive ELISA method. After a series of trials affirming the best concentration and the best time of HBsAg to cover the plate and the proper binding time of Fab to HBsAg to reach an equilibrium, we plotted the OD standard curve of the binding reaction of Fab and HBsAg using four grades of concentration of HBsAg and a series of consistency of Fab. We got the consistency of Fab at OD50% through the standard curve, and then calculated the affinity by Law of Mass Action. Results The affinity of anti-HBsAg Fab is between 10 7 -10 8 M -1 , which was only smaller by 10 times than that of anti-HBsAg IgG. Conclusion The Fab which we constructed in our lab, can strongly bind to the target antigen, thus providing a theoretical basis for its clinical use.

Objective To investigate the anticancer activity of dendritic cells(DC)transfected by recombinant adeno-associated virus(rAAV)-mediated carcinoembryonic antigen(CEA)gene in colorectal cancer treatment.Methods The recombinant plasmid rAAV/CEA was constructed and the rAAV/CEA virus stock was prepared.DC precursors were isolated from peripheral blood mononuclear cells(PBMC)by density gradient centrifugation and transfected with rAAV/CEA virus stock.The CEA expression,chromosome integration and efficiency of rAAV/CEA virus were determined by PCR,Southern blot and flow cytometery.DC precursor cells were induced by granulocyte macrophage colony-stimulating factor(GM-CSF),interleukin-4(IL-4)and tumor necrosis factor-?(TNF-?)to mature DC.The mature DC and T cells were mixed,then cultured with interleukin-2(IL-2)and interleukin-7(IL-7)resulting in generation of cytotoxic T lymphocyte(CTL),whose lethal effects on cancer cells were determined by 51chromium(51Cr)release assay.3H incorporation method was employed to detect the influence of rAAV/CEA transfected DC in the proliferation of activated T cells.Results rAAV medicated CEA gene was transferred into DC precursor successfully and the expression was high.Over 90% DC precursors expressed CEA protein.The viral gene was integrated into the DC chromosome.rAAV/CEA transfected DC could induce specific CTL which could specifically recognize and kill CEA-positive colorectal cancer LoVo cell line,and showed restrictive activity of anti-MHC class I antigen,while it was not able to kill the CEA-negative cancer cells.Conclusion The present study confirms that rAAV/CEA transfected DC vaccine can induce anti-tumor activity in vitro.It paves the way for further study on CEA as a target antigen for gene therapy on colorectal cancer.

Objective Epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of colorectal cancer (CRC). There are several potential therapies to target EGFR. The detection of EGFR expression is usually performed by immunohistochemistry (IHC) only in the primary tumor. There are a few data regarding the EGFR status in the corresponding distant metastases. The aim of the present study was to correlate EGFR expression in primary tumors and related metastases in order to find out whether EGFR status is different between primary tumors and related metastases. Methods 37 cases (25 male, 12 female) of metastatic colorectal cancer were collected. The site of primary tumor was on colon in 16 patients (43.2%) and on rectum in 21 patients (56.8%). Metastatic sites analyzed were liver in 32 patients (86.5%), inguinal lymph nodes in 5 patients (13.5%). The EGFR status of primary tumors and related metastatic sites were evaluated retrospectively. The expression of EGFR was analyzed by immunohistochemical staining (IHC), EGFR status was defined as positive if the percentage of stained malignant cells was ≥5%, and the expression feature was analyzed. Results EGFR status was positive in 25 primary tumors (67.6%), in which the corresponding metastatic site was negative in 7 cases (28.0%), whereas it was positive in 3 metastases (25.0%) from 12 cases of EGFR-negative primary cancers. The difference between the primary tumors and metastatic lesions had no statistic significance (P=0.23). Conclusion When analyzed by IHC, The difference of EGFR status has no statistic significance between the paired primary tumors and distant metastatic lesions, and 73% of samples show concordant EGFR status.

Objective To investigate the best fermentation and induction models of the engineered Escherichia coli, in order to obtain the highest expression level of humanized anti HBsAg Fab. Methods The optimal condition governing the growth of the Escherichia coli, with the flask shaking method and the best fermentation and induction conditions to yield the highest production level were explored, and then E coli were grown in fermentor using the fed batch method following the same principle under flask shaking condition to ensure the best production. Results The data obtained from flask shaking conditions showed that when the induction procedure started the amount of anti HBsAg Fab would reach the highest level at mid log growth phase under the induction condition of 25℃ and 0 2% arabinose. Using the DO stat fed batch method, the OD 600 value of the culture would reach 55 2, which corresponded to 110g/L bacterial wet weight. The biological activity of Fab was proved to have well preserved. Conclusion We established the optimal production technic of HBsAg Fab, and lay a foundation to produce HBsAg Fab on a large scale

Objective To explore the expression of Ezrin in hepatocellular carcinoma(HCC),and the relation of Ezrin expression to E-cadherin,proliferating cell nuclear antigen(PCNA) and clinical features of HCC.Methods Forty-nine cases of HCC were divided into high and low invasive groups according to tumor size,disseminated lesions,distant metastasis,tumor capsule and portal vein tumor thrombus.The expression of Ezrin,E-cadherin and PCNA in normal liver tissues,HCC and adjacent tissues were determined by immunohistochemistry,and the relation of the expression to clinical features was analyzed.Results The Ezrin expression was found in normal liver tissues,HCC and adjacent tissues,and was significantly higher in HCC tissues than in adjacent and normal liver tissues(P

Objective To compare the short-term effect of laparoscopic surgery with open surgery for gastrointesti﹣nal stromal tumor. Methods 26 patients, whose tumor were localized and the pathologic diagnosis were gastrointesti﹣nal stromal tumor in stomach after surgery, were randomly divided into laparoscopic surgery group (n=12) and open surgery group (n= 14). The age and gender level, disease duration, clinical symptom, tumor location, and the lesion diameter were similar between the two groups (P> 0.05). Surgery achievement, intraoperative blood loss, operative time, postoperative hospital stay, postoperative flatus time, postoperative liquid time, and incidence rates of postoper﹣ative complications were compared between the two groups. Results All the patients get successful R0 resection. The volume of intraoperative blood loss, operative time, postoperative hospital stay, postoperative flatus time, and postop﹣erative liquid time in laparoscopic surgery group were lower than open surgery group (P< 0.05). There was no sig﹣nificant difference in the incidence rate of postoperative complications between the two groups (P> 0.05). Conclusion Compared with open surgery, the short-term effect of laparoscopic surgery were better for patients whose gastrointestinal stromal tumor in stomach were localized, and it is worth of promoting clinical application.

Objective To compare HER-2 state in breast cancer tissue deteced by fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) and analyze their correlation. Methods HER-2/neu protein expression and gene amplification were detected by FISH and IHC in 56 newly-diagnosed cases of female breast cancer from July 2008 to July 2009. Results Of the 56 patients, HER-2 protein expression (-), (+), (++), (+++) was 9 cases (16.1%), 29 cases (51.8%), 11 cases (19.6%) and 7cases (12.5%) respectively; 26 cases (46.4%) had HER-2 gene amplification while 30 cases (53.6%) didnt have. Type of HER-2 gene amplification was mainly HER-2(++) and HER-2(+++), and according gene amplification rate was 72 7% and 100%. HER-2 (+) gene amplification rate was 37.9 %(11cases) and no gene amplification was found in HER-2(-) tissue. The HER-2 positive rate using two methods had significant difference(χ2=19.778,P<0.01). HER-2(-) and HER-2(+++) had good consistency with the FISH results(Kappa=0.969),but HER-2(+) and HER-2(+ +) were poorly consistent with the FISH results(Kappa=0.271). Conclusions IHC is the preliminary screening method for detection of HER-2 expression. HER-2(-) and HER-2(+++) have good consistency with the gene amplification, and can guide clinical treatment. Some patients with HER-2(+) and HER-2(++) have HER-2 gene amplification. FISH is needed for targeted therapy.

Objective To determine the clinical implications of internal mammary node biopsy for neoplasm stage,treatment,and prognosis in patients with breast cancer.Methods Internal mammary node biopsy via intercostal space was performed in 229 cases of breast cancer.Anatomical location of internal mammary nodes was recorded.Results Internal mammary node biopsy was successfully finished in 220 patients.There were 56 cases (24.45％ ) with internal mammary nodes metastasis,126 cases (55.02％ ) with axillary nodes metastasis,43 cases (34.13％ ) with regional metastases in both the axillary and internal mammary lymph nodes and 13 cases ( 12.62％ ) with internal mammary node metastasis only.Internal mammary node metastasis rate in patients with the number of positive axillary nodes ≥4 was 49.32％ (36/73).pN stage migration was seen in 56 patients with positive internal mammary nodes.There was no statistic relation between internal mammary nodes metastases and tumor location ( x2 =0.661,P =0.719).70.7％ patients with medial/central tumors and 50.7％ patients with the number of positive axillary nodes ≥4 were free from internal mammary node radiotherapy on account of internal mammary node biopsy.There was no complication such as pneumothorax or haemorrhagia.Conclusions Internal mammary node biopsy from intercostal space is a reliable surgical technique and can improve pN stage in some breast cancer patients.With internal mammary node biopsy,patients with a negative internal mammary node can be prevented from radiation to internal mammary nodal areas.

Objective To explore the best operation method in the patients with papillary thyroid microcarcinoma.Methods A total of 139 patients with papillary thjroid microcarcinoma were treated at our institute between Jan.2000 and Jan.2010.The data on the clinicopathological characteristics of patients and treatment were collected.Results The mean tumor size was (0.45 ± 0.24) cm.Of the 139 patients,30.2％ had multifocal tumors,19.4％ had bilateral tumors,42.4％ neck lymph node metastases.The number of eases of lymph node metastasis in level Ⅵ,Ⅱa,Ⅲ and Ⅳ was 58(41.7％),3(2.2％),10(7.9％),5(3.6％),respectively.Only one had lymph node metastasis in Level Ⅲ Microscopic extrathyroid extension was associated with neck lymph node metastases in papillary thyroid microcareinoma patients (x2 =38.39,P ＜0.001).No one developed permanent hypoparathyroidism.The median follow-up time was 103 (range,30-154) months,and local recurrence in the thyoid was diagnosed in 2 patients who underwent hemi-or subtotal thyroidectomy.Follow-up of 10 years was done in 16 cases,and the survival rate of 139 patients for 10-year was 100％.Conclusions It suggested that patients with papillary thyroid microcarcinoma has uniform clinicopathologic characteristics and the pattern of lymph node metastasis from those with papillary thyroid carcinoma.Total thyroidectomy plus level Ⅵ dissection is the optimal surgical treatment of papillary thyroid microcarcinoma.

Objective To explore the pattern of cervical nodal metastasis and the clinical significance of total thyroidectomy plus functional neck lymphadenectomy in papillary thyroid carcinoma patients. Methods Clinical and pathological data of 172 patients with papillary thyroid cancer who underwent total thyroidectomy plus functional neck lymph node dissection at Jinan Military General Hospital were retrospectively reviewed, including patient demographics, extent of surgery, parathyroid hormone level,recurrence, tumor pathology, such as tumor size, multifocality, capsular invasion, vascular invasion,extrathyroidal extension, and lymph node status. Results Of the 172 functional neck dissection patients (47 ambilateral), the incidence of lymphonodus metastasis in regions Ⅵ, Ⅳ and Ⅲ was 96. 3% ,78. 5%and 62.1% respectively. Rate of nodal metastasis was higher in patients with extracapsular invasion than in patients with no invasion ( P ＜ 0. 05 ). Serum parathyroid hormone levels significantly decreased immediately postoperatively in total thyroidectomy plus functional neck dissection and remained low for several weeks thereafter ( P ＜ 0. 01 ). The 5-, 10- and 15-year survival rate was ( 98. 83 ± 0. 82) %, (98. 23 ± 1.02 ) % and (96. 42 ± 1.43 )%, respectively. Conclusions Therapeutic neck lymph node dissection for papillary thyroid cancer is recommended for cervical nodal metastasis patients. Total thyroidectomy plus functional neck dissection is important in the treatment of papillary thyroid cancer.