Objective To explore the transfection efficiency of primary lymphocytes from human peripheral blood by different methods to acquire the method with higher transfection efficiency.Methods Mononuclear cells from human peripheral blood were isolated using Ficoll-Hypaque.Cell viability was detected by Trypan blue staining.Suspending lymphocytes were sucked out and were incubated in 24-well plate after cultured in 6-well plate for 2 h.Activated lymphocytes were transfected by electroporation with plasmid(PEGFP-N1).Resting or activated lymphocytes were transfected by lentivirus vector(LVGFP) single infection or repeated infection,respectively.Green fluorescence protein (GFP) was detected under the fluorescence microscopy and percentage of positive cells was checked by flow cytometry at different time points after infection.At the same time,the effectiveness of lentivirus infection was compared under different conditions.Results Purity of mononuclear cells isolated by Ficoll-Hypaque was 95 ％ and its viability was over 95 ％.The percentage of lymphocytes obtained with a uniform shape was 90 ％-95 ％.Scattered fluorescence was observed by electroporation under the conditions of voltage 2 100 V,pulse width 10 ms,pulse number 1 for lymphocyte,while fluorescent became weaker over time and no green fluorescent was observed after transfection for 72 h.After resting lymphocytes were infected once for 48 h by lentivirus vector,green fluorescent was not found and positive cells were less than 1％.1％-5 ％ of activated lymphocytes could express GFP after single lentivirus infection and the expression levels were enhanced with concentration increasing,while 5 ％-10 ％ of activated lymphocytes showed strong green fluorescent by repeated lentivirus infection.In contrast with electroporation,the fluorescent with lentivirus infection was stronger over time.Conclusion Repeated lentivirus infection could efficiently transfect exogenous genes into activated lymphocytes for stable expression.

Objective To evaluate the ability of the artificial pneumothorax CT examination in detection and diagnosis of pleural metastatic tumor.Methods A retrospective study of 14 patients with pleural effusion using artificial pneumothorax CT examination was conducted.Results Six of the 14 patients were found to have a nodule or nodules on the pleura.The pleural metastatic tumors of 3 cases were from the lung cancers and another 3 from breast cancers were proved histologically.Conclusion The artificial pneumothorax CT examination is a effective and simple method in detecting of pleural metastatic tumor.

Objective To evaluate and compare spiral CT and positron emission tomography-CT (PET-CT) in characterization of of axillary lymph nodes in breast cancer patients.Methods Forty patients with pathologically proven breast Cancer underwent contrast-enhanced spiral CT of tbe breast and axilla,13 of them also underwent PET-CT examination.One hundred and fifty-eight axillary lymph nodes were found in the 40 patients through contrast enhanced spiral CT,while 57 lymph nodes were found in the 13 patients through PET-CT.Three radiologists rated the lymph nodes found in CT images on a five-point scale.If the score was equal to or greater than 3,it was defined as positive (metastatic),otherwise negative.Visual observation and semiquantitative analysis were used to classify lymph nodes in PET-CT images.The results of spiral CT observation and PET-CT observation of lymph nodes were compared with pathological results.The relative value of CT and PET-CT was analyzed.Exact probability statistics were employed.Results One hundred and fifty eight lymph nodes of 40 patients were detected by spiral CT,91 of them were diagnosed as positive and 67 as negative Among the lymph nodes found in spiral CT,99 were positive and 59 were negative pathologicall.A total of 57 lymph nodes were found by PET-CT.Thirty-nine of them were defined as positive and 18 as negative.Among the lymph nodes found in PET-CT,39 were positive and 18 were negative pathologically.The sensitivity,specificity,accuracy,positive and negative predictive values in CT prediction in axillary lymph nodes metastases were 88.89%,94.91%,91.14%,96.70%,and 83.58%,respectively.The sensitivity,specificity,accuracy,positive and negative predictive values in PET-CT prediction in axillary lymph nodes metastases were 97.44%,94.44%,96.49%,97.44%,and 94.44%,respectively.PET-CT had no significant difference with spiral CT in sensitivity,accuracy,positive and negative predictive values for detection of axillary lymph nodes in breast cancer.But there was significant difierence between PET-CT and CT in negative predictive value(P＜0.05).Conclusions Both helical CT and PET-CT were the efficient methods in predicting the axillary lymph node status in breast cancer patiens.The negative predictive value of PET-CT was higher than that of helical CT.PET-CT has a better predictive ability than CT for the presurgical evaluation for breast cancer patients.

Objective To explore expression and clinical value of VEGF-C and p63 in early esophageal carcinoma and intraepithelial neoplasia. Methods 146 cases were randomized into normal esophageal mucosa, low level intraepithelial tumor, high level intraepithelial tumor and early esophageal carcinoma. The expression of VEGF-C and p63 were detected by using the immunohistochemistry dyeing.Results The expression of VEGF-C immunohistochemistry dyeing had statistical differences among different levels(X~2= 47.455, P <0.001). Normal esophageal mucosa v.s. high level intraepithelial tumor (X~2=36.721, P <0.001), Normal esophageal mucosa v.s. early esophageal carcinoma (X~2=26.483, P <0.001), low level intraepithelial tumor v.s. high level intraepithelial tumor(X~2= 10.025, P<0.0083), low level intraepithelial tumor v.s. early esophageal carcinoma(X~2=16.734, P<0.001). There was a significant correlation between pathological classification and the expression amount of VEGF-C (r = 0.462, P <0.001). The expression of p63 had statistical differences among different levels(X~2=28.962, P <0.05). There was a significant difference on normal esophageal mucosa comparing with low level, high level intraepithelial tumor or early esophageal carcinoma (X~2=12.735, P =0.005, X~2=20.421, P<0.001, X~2=20.854, P<0.001). There was a significant correlation between pathological classification and the expression of p63 (r= 0.272, P<0.05). Conclusion There is a significant correlation in the express of either VEGF-C or p63 comparing with either intraepithelial tumor or early esophageal carcinoma. It may be an early warning indicator.

ObjectiveTo explore the relationship between chronic kidney disease (CKD) and cerebral small vessel disease (SVD) in elderly patients. MethodsOne hundred and fifty-two elderly male CKD patients for experimental group and 158 elderly male for control group were recruited. Demographic data and vascular risk factors were recorded. White matter lesion (WML) was semi-quantitatively assessed by cerebral magnetic resonance imaging (MRI), and lacunar infarction (LI) was also calculated. Results(1) The prevalenees of hypertentsion and diabetes mellitus were higher in elderly CKD patients than those in control group (30. 9% vs. 19.0%, 23.7%vs. 14.6%;both P～0. 05). (2) The percentages of grade 2 and grade 3 WMLs were higher in elderly CKD patients than those in control group (34.9% vs. 24.1%, 25.7% vs. 16.5%;both P<0.05). Prevalence of LI was higher in elderly CKD patients than that in control group (45.4% vs.25.3% ,X2= 13. 70, P<0. 05). The similar Resultswere also obtained except for control subjects with hypertension and diabetes mellitus. (3) The logistic regression analysis showed that age, hypertension and low glomerular filtration rate (GFR) were closely associated with SVD in elderly CKD patients. ConclusionsHypertention and diabetes mellitus are risk factors for CKD in elderly patients. SVD is associated with CKD, and age, hypertension and low GFR may be risk factors for SVD in elderly CKD patients.

Objective To study the safety management and risk response of preoperative emergency in patients with cerebral hemorrhage. Methods 92 patients with intracerebral hemorrhage were divided into two groups. The conventional group was treated with routine care in prehospital first aid. The prehospital intervention group used transport safety management and risk comprehensive response measures. The incidence of first aid consciousness,rapid response score, safe transshipment score, medical care score and the success rate of rescue and the incidence of medical disputes were compared between the two groups. The satisfaction of the family members of the patients with first aid treatment was analyzed. Results The score of first aid consciousness, rapid response score, safe transfer score,medical care score of the prehospital intervention group were (96. 24 ± 3. 91) points,(93. 98 ± 2. 72) points, (95. 26 ± 3. 57) points and (95. 72 ± 3. 02) points,respectively,which were significantly better than those of the conventional group [(83.12 ±3.15)points,(82.02 ±1.53)pointss,(83.52 ±2.92)points,(83.14 ±2.15)points, t=8. 629,8. 826,8. 042,8. 514,all P <0. 05]. The success rate of hospitalization of the prehospital intervention group was 95. 65%,which was higher than 78. 26% of the conventional group(χ2 =6. 133,P<0. 05). The incidence rate of medical disputes in the prehospital intervention group was significantly lower than that in the conventional group (χ2 =5. 145,P<0. 05). Among them,there were 9 cases of medical disputes in the conventional group,the incidence rate was 19. 57%. In the prehospital intervention group,there was 1 case of medical disputes,the incidence rate was 2. 17%. The satisfaction rate of prehospital emergency treatment in the prehospital intervention group was 95. 65%, which was significantly higher than 78. 26% in the conventional group (χ2 =6. 133,P<0. 05). Conclusion The safety management and risk response measures of preoperative emergency in patients with intracerebral hemorrhage are effective,which can effectively improve the success rate of cerebral hemorrhage rescue,improve nursing staff first aid consciousness and rapid response ability,improve medical care and transport safety,it is worthy of promotion.

Objective To investigate the feasibility of Wilms’tumor gene 1 (WT1)-specific CD8+T cells from periph?eral blood for the treatment of breast cancer by detecting the killing activity of WT1 specific CD8+T cells on breast cancer cells. Methods Flow cytometry was used to detect WT1-specific CD8+T cells in the peripheral blood of 20 samples from HLA-A2 seropositive healthy donors, which were isolated by WT1/MHC streptamer magnetic beads and cultured. The func?tion of WT1-specific CD8+ T cells were analysis by cytotoxicity assay. Results Twelve of 20 healthy donors had naive WT1-specific CD8+T-cell frequencies of>0.5%, and 4 of 20 even>1.0%of all CD8+T cells. After positive selection by magnetic cell separation, a purity of up to 80%can be achieved. WT1 specific CD8+T cells can specifically kill breast can?cer cell line with WT1 polypeptide. Conclusion WT1 specific CD8+T cells can be detected in peripheral blood of healthy volunteers. WT1 specific CD8+T cells have killing effect on breast cancer cells, suggesting the feasibility of adoptive immu?notherapy for breast cancer.

This study was aimed to observe the influence of Discornin Tablets on activation nuclear transcription factor NF-κBp65 of rheumatoid arthritis (RA) cell model as well as the expression of MMP-9, VEGF and tumor necrosis factor-α (TNF-α). Interleukin-17 (IL-17) and TNF-α were used for stimulating RSC-364 cells. Discornin Tablets at different concentrations were used for intervention. The influence of Discornin Tablets in different concentrations on cell viability was detected by MTT method. Expressions of NF-κBp65 and its inhibitory protein (IκB-α) in each group were detected by western blot method. Changes in VEGF, MMP-9 and TNF-α protein levels in cell broth supernatant were checked by ELISA. The results showed that Discornin Tablets can promote the expression of κB inhibitory pro-tein, reduce the high expression of NF-κB protein level, and inhibit the cellular secretion of VEGF, MMP-9 and TNF-α. It was concluded that Discornin Tablets had negative regulation effect on nuclear transcription factor κB of RSC-364 cells. It can increase the expression of IκB-α, as well as reduce the secretion of inflammation factors and blood vessel newborn factors. It suggested that Discornin Tablets may have the potential regulation effect on RA.

Objective To investigate the effects of B lymphocyte-induced maturation protein-1 ( Blimp-1) on the number and function of splenic lymphocytes.Methods The mice with defective Blimp-1 in T cells were generated by cross-breeding B6.Blimp-1flox/flox mice with B6.Lck-Cre mice.The mononuclear lymphocytes isolated from spleen of T cell conditional Blimp-1 knockout (Blimp-1CKO) mice and wild type ( WT) C57/B6 mice were comparatively analyzed.Alterations of CD4+T and CD8+T cell subsets, the secre-tion of cytokines as well as the expression of C-C chemokine receptor type 7 ( CCR7 ) and Sphingosine-1-phosphate receptor 1 (S1P1) in mice from the two groups were analyzed by flow cytometry.The changes of CD19+B cell subsets were also detected.Results Compared with WT mice, the total numbers of mononu-clear cells, T and B lymphocytes were all significantly increased in Blimp-1CKO mice ( P<0.05) .The ab-solute numbers of CD4+T, CD8+T and CD19+CD5+CD1d+B cells in mice form Blimp-1CKO group were higher than those of the control group (P<0.05), however, no significant differences with the percentages of these cell populations were observed between two groups.Higher numbers and percentages of CD19+CD5+B cells were detected in mice from Blimp-1CKO group (P<0.01).The Blimp-1CKO mice showed increased secretion of IFN-γ, TNF-α, IL-17 and IL-2, but decreased expression of CCR7 on CD8+T cells as com-pared with WT mice (P<0.05).No significant differences with the changes of S1P1 were found between the two groups.Conclusion Blimp-1 played an important role in the maintenance of number, phenotype and function of T cells.Furthermore, not only T cells but also B cell subsets in mice were affected by the dele-tion of Blimp-1 in T cells.