Abstract:Objective：To construct over-expressing pCAG-3×flag-PP2A Cα transgenic mice，screen the stable system of high level expression，and therefore to provide a transgenic animal model for the study of the biological function of PP2A Cα. Methods：The pCAG-3×flag-PP2A Cα transgenic vectors were constructed by inserting mice PP2A Cα cDNA and Flag protein sequence tag to the downstream of pCAG promoter. The linear pCAG-3×flag-PP2A Cα plasmid was injected into the zygote nucleus by microinjection technique to get the over-expressing pCAG-3×flag-PP2A Cα mice. The positive over-expression mice were identified by PCR. The positive expression of total protein in mice was extracted and the expression of transgenic mice was analyzed at the protein level. Results：The pCAG-3×flag-PP2A Cα transgenic vector and transgenic mice was successfully constructed by PCR and was verified by plasmid sequence analysis. The system of high level expression transgenic mice was screened by Western blot assay. Conclusion：The pCAG-3×flag-PP2A Cα transgenic mice with stable system of high level expression were screened and obtained.

Abstract:Objective：To develop monoclonal antibodies（mAb）against human neuropilin 1（HuNRP1）and provide useful materials to inhibit tumor angiogenesis by targeting HuNRP1. Methods：Using lymphocyte hybridoma technique，8-week-old BALB/c mice were immunized with recombinant protein，b domain of human neuropilin 1（TF-HuNRP1b）derived from E. Coli，then splenocytes of the immunized mice were prepared and collected，with the addition of sp2/0 myeloma cells，all the mixed cells were fused. Positive hybridoma cells were screened by indirect immunofluorescent assay（IFA）established with native HuNRP1 expressed by breast tumor cell line MDA-MB-231. The specificity of mAb was identified by IFA，Western blot and flow cytometry（FCM）. The inhibitory ability of mAbs against tumor angiogenesis was identified by methyl thiazolyl tetrazolium（MTT）and chamber migration test. Results：One hybridoma cell line producing mAb against TF-HuNRP1b were obtained and named as 4F11. Western blot analysis showed that mAbs could recognize only recombinant HuNRP1b not the other proteins produced in BL21（DE3）（pCold-HuNRP1b）and BL21（DE3）（pCold）. The results of IFA and FCM showed that mAb could recognize native HuNRP1 expressed on human umbilical vein endothelial cells（HUVEC），tumor cell line MDA-MB-231 and HepG2. The results of MTT assay and chamber migration test further showed that mAb 4F11 had strong inhibitory effect on migration and proliferation of HUVEC. Conclusion：mAb against HuNRP1b was successfully obtained which will be useful in the functional study of HuNRP1 and provide good materials for the inhibition of tumor angiogenesis.

Abstract:Objective：To establish stable and efficient method for primary culture of human palatine tonsil crypt epithelial cells. Methods：Eighty palatine tonsil tissues were derived from children aged from 3 to 5 undergoing surgical removal of tonsil. Tissue piece method and combined type Ⅱ dispase and 0.25% trypsin-EDTA digestion method were used to separate primary palatine tonsil crypt epithelial cells，and the effectiveness of two isolation methods were compared. Serum-free Keratinocyte medium were applied to purification and primary cultivation. Growth feature and morphology of primary cells were observed under an inverted microscope. Immunofluorescence and immunocytochemistry technique were used to identify the specification of primary cell. Results：The success rate，density and primary cell fusion time of the dispase digestion group were all higher than those of the tissue piece group（P < 0.05）. The primary tonsil crypt epithelial cell began adherent growth after 2 days isolation from the tonsil tissues. The shape of cell was polygon under microscope，and cells connected like islands. Primary cells formed confluent monolayers after 12 days’ cultivation，it seemed like paving stone. Pancytokeratin positive staining was determined by immunocytochemistry. The specific cytokeratin 8/18 staining was positive after immunofluorescence identification. Conclusion：It is an efficient and repeatable method for primary isolation and cultivation of human palatine tonsil crypt epithelial cells by using combined type Ⅱ dispase and 0.25% trypsin-EDTA digestion method with serum-free keratinocyte medium.

Abstract:Objective：To investigate the expression of SPOCK2 gene in hyperoxia-induced bronchopulmonary dysplasia（BPD）models of neonatal rats. Methods：BPD models were induced by 85% O2 exposure. Newborn SD rats were randomly divided into the air group and the hyperoxia（85% oxygen）group. The pathological changes of pulmonary tissues were observed by hematoxylin-eosin（HE）staining. The expressions of SPOCK2 mRNA were detected by real-time quantitative PCR. The expression of SPOCK2 protein was detected by immunohistochemistry. Meanwhile，A549 lung epithelial cells were stimulated by hyperoxia in vitro and the expression levels of SPOCK2 mRNA were detected by qPCR. Results：BPD neonatal rat model was successfully constructed. The pathological changes of BPD were found in the pulmonary tissues of the model group by HE staining. The value of pulmonary radical alveolar counts（RAC）in the model group was significantly lower than that of the control group at 10 d and 14 d after birth（P < 0.05）. As shown with immunohistochemistry，the expression of SPOCK2 protein in the lung tissues of rats in the control group was higher than that in the model group，especially at 10 d and 14 d after birth. The expression of SPOCK2 mRNA in the lung tissues of rats in the control group was higher than that in the model group，which reached its peak on 18 d after birth and decreased in adulthood. Compared with the control group，the expression of SPOCK2 mRNA in the model group decreased. There were significant difterences befween two groups at 10 d and 14 d after birth（P < 0.05）. The expression of A549 mRNA in the model A549 cells was higher than that in the control（P < 0.05）. Conclusion：SPOCK2 gene may be involved in pulmonary development process，and may protect lung tissue during hyperoxia stimulation.

Abstract:Objective：To observe the protective effects and mechanisms of hydrogen sulfide（H2S）on isoproterenol（ISO）induced acute myocardial injury in mice. Methods：C57BL/6 mice of 12-week-old were subcutaneously injected with H2S donor NaHS［10 μmoL/（kg·d）］for 2 weeks，and then large dosage of ISO［150 mg/（kg·d）］was jointly given by subcutaneous injection for 2 days. Pathological structures of myocardium were assessed. Activities of creatine kinase（CK）and lactate dehydrogenase（LDH）in plasma were measured. Levels of reactive oxygen species（ROS）and malondialdehyde（MDA），total antioxidant capacity（T-AOC），activity and expression of superoxide dismutase（SOD）in myocardium were detected. Results：NaHS pretreatment improved ISO induced pathological disorder in the myocardium，reduced CK and LDH activity in the plasma，decreased the level of myocardial ROS and MDA，improved myocardial T-AOC，enhanced myocardial SOD activity and expression. Conclusion：H2S alleviated ISO induced acute myocardial injury in mice，which might be associated with the improvement of SOD expression and activity，and the reduction of oxidative stress.

Abstract:Objective：To establish a chronic renal failure（CRF）rat model and to investigate the effects of prostacyclin（PGI2）derivatives on the expressions of key factors in renal local renin angiotensin system（RAS）of CRF rats，including angiotensin Ⅱ（AngⅡ），angiotensin（1-7）［Ang-（1-7）］，angiotensin converting enzyme 2（ACE2），and angiotensin Ⅱ type 1 receptor（AT1R），thereby to explore the potential protective mechanism of prostacyclin derivatives on CRF. Methods：A total of 30 SD male rats were divided into the sham operation group and the operation group at a ratio of 1∶2. Rats in the operation group underwent 5/6 nephrectomy followed by biochemical and pathological examinations 5 weeks after the surgery to determine the success of modeling，and then the operation group was further randomized into the model group and the treatment group at a ratio of 1∶1. Rats in the treatment group were given beraprost sodium（BPS），a PGI2 derivative at a dosage of 0.6 mg/（kg·d） through two intragastric administrations from the fifth week after operation，and those in the model group and the sham operation group received same volume of distilled water. After 4 weeks of treatment，serum creatinine（Scr）and urea nitrogen（BUN）were detected，and 24-hour urine protein excretion（24 hUPE）was measured. The rats were then sacrificed and pathological changes of renal tissues in rats were observed by HE and Masson staining. Expressions of ACE2 and AT1R in renal tissues were detected by real-time quantitative polymerase chain reaction（qRT-PCR），Western blot and immunofluorescence，and enzyme linked immunosorbent assay（ELISA）was employed to measure the expressions of AngⅡ and Ang（1-7）in kidey and ACE2，AngⅡ，Ang（1-7）and AT1 in serum. Results：24hUPE，Scr and BUN levels were higher in the operation group at 5 weeks and 9 weeks than those in the sham group，with statistical significance. Compared to levels at 5 weeks，Scr and BUN were remarkably decreased in the treatment group at 9 weeks，significantly different from those in the model group with mild reduction，but still higher than those of the model group at 9 weeks，whereas urine protein changed in the opposite direction. Renal pathology showed mesangial cell proliferation and matrix hyperplasia in the operation group，accompanied with changes such as renal interstitial inflammatory cell infiltration，interstitial fibrosis and glomerular sclerosis，on the contrary，above changes were improved in the treatment group. AngⅡ and AT1R in the kidney of the operation group were up-regulated than those in the sham operation group，but Ang（1-7）and ACE2 were down-regulated at the same time. Meanwhile，there were statistical differences in changes of the four factors in RAS in the treatment group，yet no such changes in the kidney were found in the serum. Conclusion：Prostacyclin derivatives reduce the 24 hUPE，relieve the chronic renal fibrosis，and possibly delay the progress of chronic kidney disease via a potential mechanism of regulating the expressions of key factors AngⅡ，ACE2，Ang（1-7） and AT1R in renal local RAS system in the CRF rat model.

Abstract:Objective：To study the influence on pulmonary fibrosis and expression of Bax/Bcl-2 in the lung after blocking interleukin（IL）-17 activity in mice induced by bleomycin（BLM）. Methods：A total of 80 C57BL/6 mice were randomly divided into the following 4 groups，including the model group treated with neutralizing IL-17 antibody，the model group treated with isotype-matched control antibody，the model group and the PBS group. The three model groups were received a single intratracheal instillation of 5 mg/kg body weight of BLM in 0.05 mL sterile saline，while the PBS group were administrated the equivalent sterile saline. All mice were injected from the caudal vein with neutralizing rat antimouse IL-17 mAb，or control rat IgG，or PBS alone every 3 d starting on d1 before making models. At 28 d after model establishment，lung tissues from all mice were removed and used to measure the extent of pulmonary fibrosis by Masson staining and hydroxyproline contents measurement. The apoptosis rate of pulmonary cells was detected by flow cytometry（FCM）. Meanwhile，the expressions of Bax/Bcl-2 in mice were also evaluated by immunohistochemistry（IHC）. Results：Compared with other groups，the pulmonary fibrosis degree，hydroxyproline contents and apoptosis rate were significantly decreased in mice blocked with anti-IL-17 mAb（P < 0.01，respectively）. Bax protein expression was decreased obviously（P < 0.01），although the Bcl-2 protein expression had no obvious changes，but the ratio of Bax/Bcl-2 decreased significantly（P < 0.01）. Conclusion：Pulmonary fibrosis had been improved significantly after the endogenous IL-17 activity of mice blocked，the apoptosis rate and the ratio of Bax/Bcl-2 were all decreased in lung tissues. These data suggested that IL-17 improving the pulmonary fibrosis induced by BLM may be associated with Bax/Bcl-2 mediated mitochondrial apoptotic pathways.

Abstract:Objective：To investigate the value of microRNA-122（miR-122）in the evaluation of hepatotoxicity at the early stage. Methods：Based on mouse primary liver cell injury models induced by acetaminophen，the concentrations of miR-122 and traditional cytotoxic marker lactic acid dehydrogenase（LDH）in the cell culture supernatant were measured parallelly，and followed by the correlation analysis. Besides，multiple cellular models induced by different potential hepatotoxic substances were established，and the concentrations of miR-122 and LDH in supernatant were analyzed and compared. The advantage of miR-122 for hepatotoxicity evaluation was further verified. Results：There were well correlations between miR-122 and LDH for hepatotoxicity evaluation（r > 0.9）；Moreover，miR-122 demonstrated higher sensitivity compared with LDH. Conclusion：miR-122 has potential application value as a biomarker for early evaluation of drug hepatotoxicity.

Abstract:Objective：This research was aimed to develop a single-tube multiplex assay of 28 autosomal SNPs to validate its ability of differentiating population from 5 continents and estimate its value in cases. Methods：We selected 28 SNPs from The Global AIMs Nano set to develop the single-tube multiplex assay using SNaPshot technique system. Using this system，we detected 712 individuals from 16 populations in our lab. We combined these results and data that collected from 1 000 genomes and HGDP-CEPH（total 2 804 samples，38 populations）together and used structure cluster analysis and frequency PCA analysis to evaluate this panel. We selected samples of which ancestry component was greater than 90% to build classification library of reference population. Analyses of group matching probability（MP） and individual principal component were performed in 140 individual samples to evaluate the differentiation ability on population source of the multiplex assay in actual samples. Results：Not only could this multiplex assay of 28 SNPs differentiate the population from 5 continents but the admixture of South Asia and Eurasian population. The ancestry component and MP value of some known origin samples were consistent with the sample information. Conclusion：This multiplex assay can differentiate the population from 5 continents and differentiate the ancestry origins of individuals correctly. This multiplex assay is applicable for criminal investigations in the forensic practice.

Abstract:Objective：To investigate the association between serum lipoprotein-associated phospholipase A2（LP-PLA2）and coronary atherosclerosis. Methods：A total of 119 inpatients from the Second Affiliated Hospital of NMU were selected in this study. Eighty-three patients with coronary atherosclerosis were included in the coronary lesion group，and 36 people without coronary atherosclerosis were included in the negative group. LP-PLA2 was detected by enzyme kinetic method，high sensitive C-reaction protein（hs-CRP）was detected by enzyme linked immunosorbent assay（ELISA），and the level of D-Dimer was detected by immune turbidimetry. The patients in the case group were divided into three groups including 1 branches，2 or 2 branches lesion + left main lesion，and 3 or 3 branches lesion according to the number of diseased coronary artery. The severity of coronary artery lesion was evaluated by the Gensini integrations. Results：The serum levels of LP-PLA2，hs-CRP and D- Dimer in the case group were significantly higher than those in the control group（P < 0.05）. The levels of LP-PLA2 and hs-CRP were significantly different between patients with different severity of coronary lesions（P < 0.05）. Furthermore，Pearson correlation analysis showed that LP-PLA2 and hs-CRP had a low-level positive correlation with Gensini score（r=0.418，r=0.365，P < 0.05）. Conclusion：The levels of serum LP-PLA2，hs-CRP and D-Dimer were correlated with coronary atherosclerosis. For patients with coronary atherosclerosis，serum levels of LP-PLA2 and hs-CRP can only provide a limited reference value in assessing the extent of coronary artery lesions.

Abstract:Objective：To explore the risk factors of blade excessive lateral migration among femoral intertrochanteric fracture fixed with proximal femoral nail anti-rotation（PFNA）. Methods：A total of 163 intertrochanteric fracture patients（mean 69.9 years old）who received PFNA fixation from April 2011 to March 2016 in the Affiliated Hospital of Jiangsu University were retrospectively evaluated. There were 84 male patients and 79 female patients aged from 50 to 99 years（mean age of 69.6 years）. The radiological parameters were measured，including the condition of proximal lateral wall（broken or not），the position of lesser trochanter（displaced or not），tip-apex distance（TAD），calcar referenced tip-apex distance（Cal-TAD），the amount of lateral migration of the blade and medial cortex support mode. Singh index and AO/OTA type were also recorded. Logistic regression model was established to analyze the key factors causing excessive lateral sliding of blade. Results：We found that 18 cases showed radiographic over lateral migration. The univariate analysis illustrated that the excessive lateral migration of blade directly related to unstable fracture type，displaced lesser trochanter，and negative medial cortex support（P < 0.05），but not Singh index，calcar reduction，lateral wall injury，TAD，CalTAD，age and gender（P＞0.05）. Multiple logistic regression suggested that unstable fracture pattern and negative medial cortex support were independent risk factors of blade excessive lateral migration（P < 0.05）. Conclusion：For femoral intertrochanteric fracture patients treated with PFNA，over lateral sliding of blade may occur. The risk factors include unstable fracture pattern and negative medial cortex support. PFNA should be used cautiously in those with the hazard factors，and weightbearing ambulation must be delayed.

Abstract:Objective：To explore clinical and immune function effect of spleen polypeptide injection combination with concurrent chemoradiotherapy in patients with limited-stage small cell lung cancer（LS-SCLC）. Methods：From January 2013 to January 2017，76 LS-SCLC patients were divided randomly into observation and control groups（38 patients in each）from the Affiliated Cancer Hospital of Nanjing Medical University. Control group were given concurrent chemoradiotherapy，and observation group were given the same therapy combined with spleen polypeptide injection. The clinical results，T cell and its subtypes，survival probabilities and adverse reactions were evaluated. Results：The improvement rates of Karnofsky performance score（KPS）in the observation group were significantly better than those in the control group（P < 0.05）. The observation and control groups had a similar response rate（84.2% and 89.5%，respectively）. When comparing hematologic toxicity，the observat group had a less incidence rate of leucopenia（60.5% vs. 84.2%，χ2＝5.33，P=0.04）. Compared with pre-treatment，the levels of CD3+，CD4+and CD4+/CD8+ in the observation group were increased and had significant differences（P < 0.05），whereas，the control group had no obviously increasing. The progress-free survival of observation group was significantly improved comparing with that in control group（P=0.03）. However，there was no statistic significance in the overall survival between two groups（P=0.22）. Conclusion：Spleen polypeptide injection can improve the activity of immune function and prolong PFS of the patients during the chemoradiotherapy for LS-SCLC.

Abstract:Objective：To explore the variation of hand-foot-mouth disease（HFMD）in China from 2008 to 2014 and provide implements for preventing the disease. Methods：The HFMD data from 23 provinces，5 autonomous regions and 4 municipalities was obtained from National Center for Disease Control and Prevention according to month. Standard deviation ellipse，median center and hot-spot analyses were employed to analyze HFMD data. Results：①During the year 2008 to 2014，it was reported 11 814 830 HFMD cases，with an average growth rate of 125.55 per 100 thousandth.②The outbreak of HFMD was in April to July and September to November，but different regions had different characteristics.③There were higher HFMD incidence rate in southeastern areas with double peaks than that in northwestern areas with single peak.④HFMD incidence rate had an increasing trend in southern areas. Conclusion：There was a dominantly regional difference at period of incidence and diffusion. Thus，different provinces should implement prevention and control measures to reduce the incidence and mortality depended on its characteristics.

Abstract:Objective：To evaluate the effect of atrial fibrillation，stroke and case fatality rate in elderly patients（age ≥80 years）undergoing on-pump coronary artery bypass surgery（CCAB）or off-pump coronary artery bypass surgery（OPCAB）. Methods：We searched relevant literature in MEDLINE，EMBASE，Cochrane Library. Meanwhile，conference records or gray literature from cardiovascular congresses were collected to compare CCAB and OPCAB for elderly patients. Meta analysis was performed using Review Manager 5.3 software. Results：A total of 15 articles were included in the meta-analysis，all of which were observed，and randomized controlled studies（RCT）were not retrieved. A total of 3 271 patients were enrolled in this meta-analysis，of which 1 573（48%）were CCAB. Meta-analysis showed that the OPCAB significantly reduced the incidence of postoperative patients with stroke（OR = 2.77，95% CI：1.65~1.65，P < 0.001）and case fatality rate（OR = 2.08，95% CI：1.52~1.52，P < 0.001），and the differences were statistically significant（P < 0.05）. Two groups of patients with had no significant difference on postoperative incidence of atrial fibrillation（OR = 1.01，95% CI：0.84~0.84，P=0.91）. Conclusion：Compared with CCAB，OPCAB can significantly reduce the incidence of postoperative stroke and case fatality rate in elderly patients，but big size randomized controlled trials are needed for further verification.

Abstract:Objective：To systematically evaluate the intervention effect of health education on prevention of mother to child transmission of acquired immune deficiency syndrome（AIDS） in childbearing women in China. Methods：We retrieved full text databases at home and abroad by computer and collected relevant literature on the health education in preventing mother-to-child transmission of HIV among childbearing women in China from January 1，2010 to December 31，2015. Rev Man 5.0.23 software and meta analysis method were performed to comprehensively quantitatively analyze 19 included studies. Results：After health education intervention，the total awareness rate of prevention of mother to child transmission of HIV among childbearing women in China increased by 41%（Z=7.84，P<0.001），the rate of the correct attitude held increased by 22%（Z=4.08，P=0.008），and the positive behavior change rate increased by 27%（Z=5.12，P < 0.001）. Subgroup analysis showed that the effect of health education in low baseline was better than that in high baseline（χ2=68.31，P < 0.001），the effect of positive health education was better than that of general health education（χ2=40.96，P < 0.001），and the effect of health education with high quality research and low quality research was similar（χ2=0.29，P=0.840）. Conclusion：Health education intervention has significant effect on improving the awareness rate of knowledge in preventing mother-to-child transmission of HIV，the correct attitude and behavior change rate of childbearing women in China.