Abstract:Objective: To produce Trop-2-targeted chimeric antigen receptor T cells(CAR-T cells) of human trophoblast cell surface antigen 2(Trop-2) and detect their effects on the proliferation of ovarian cancer cells in vitro. Methods: Molecular cloning and gene recombination technology were introduced to construct Trop-2 CAR vector. The expression of Trop-2 CAR on 293T cells was tested by Western blot. The effect of Trop-2 CAR-T cells on the proliferation of ovarian cancer cells was evaluated by CCK-8 assay. The secreting changes of cytokines by Trop-2 CAR-T cells were detected by ELISA. Results: The results of enzyme digestion and sequencing demonstrated that the Trop-2 CAR vector was constructed correctly. The results of Western blot showed that Trop-2 CAR was expressed on 293T cells. The results of CCK-8 verified that Trop-2 CAR-T cells prominently inhibited the growth of ovarian cancer cells with Trop-2 antigen expression(P<0.05), moreover, higher levels of IFN-γ and IL-2 were detected in supernatant by ELISA(P<0.01). Conclusion: Trop-2 CAR-T cells were prepared successfully and could inhibit the proliferation of ovarian cancer cells expressing Trop-2 antigen.

Abstract:Objective: To construct a UV-responsive human epidermal growth factor receptor 2 (HER-2)-targeting immunoliposome, and investigate the improvement of chemotherapy efficacy on breast cancer. Methods: We fabricated the trastuzumab Fab fragment-decorated immunoliposome by thin film-dispersion method, which formed intermolecular cross-linking through the diacetylenic group by ultra-violet (UV) irradiation. The liposomal morphology was observed with transmission electron microscope. The particle size distribution and serum stability were evaluated by dynamic light scattering. Flow cytometry was performed to compare the intracellular uptake of liposomal and free drugs. The in vitro and in vivo anti-tumor efficacy of liposomal drugs against MCF-7 cells were evaluated by CCK-8 and subcutaneous tumor inhibition analysis, respectively. Results： After UV irradiation, the immunoliposome exhibited a fine spherical structure, better serum stability and slower drug release with a decreased mean diameter of (88.1±13.2) nm. The cellular uptake of immunoliposome(PC-DOX-Fab) was significantly enhanced comparing with non-targeting liposomes(PC-DOX-BSA) and free drug(DOX)(P<0.05). Cytotoxicity assays against HER-2+ cancer cells showed that the tumor suppressing efficacy of immunoliposomal drugs was significantly improved comparing with PC-DOX-BSA and free DOX at the same condition(P<0.05). In vivo studies demonstrated that the treatment of PC-DOX-Fab significantly reduced the tumor growth compared with that of PC-DOX-BSA and free DOX (P<0.05). Conclusion: In this study, we identified a novel liposomal drug delivery system for improved chemotherapy against HER-2-positive breast cancer cells, which owned a fine spherical structure and excellent serum stability. The in vitro and in vivo experimental results clearly suggested that this UV-responsive immunoliposome exhibits potent tumor suppressing activities, which deserved further investigation for clinical application.

Abstract:Objective：To explore the roles and the potential underlying molecular mechanisms of Osterix(Osx) on the migration and invasion of breast cancer cell line MDA-MB-231. Methods: Real-time PCR and Western blot assay were performed to detect the expression levels of lysyl oxidase (LOX) in Osx stably transfected or knocked down cells as well as in their corresponding control cells (231-OE6, 231-OEC, 231-KD2, and 231-KDC). The migration and invasion of 231-OE6 cells transfected with LOX siRNA and 231-KD2 cells transfected with LOX expression plasmids were detected by Transwell assays. Results: Compared to their corresponding control cells, the expression of LOX was increased in 231-OE6 cells, and decreased in 231-KD2 cells(P<0.05). Knockdown of LOX in 231-OE6 cells attenuated the increased migration and invasion. Conversely, overexpression of LOX in 231-KD2 cells rescued the decreased migration and invasion(P<0.05). Conclusion: The expression of LOX is up-regulated by Osx in human breast cancer cells, and it promotes the migration and invasion of breast cancer cells.

Abstract:Objective: To investigate the expression of long non-coding RNA（lncRNA） prostate cancer associated transcript 4（PCAT4） and its clinical significance in prostate cancer. Methods: Real-time-PCR method was used to determine the relative expre-ssion of PCAT4 in 48 cancer samples and corresponding paracancerous tissues from patients with prostate cancer, as well as prostate cancer cells PC3, 22RV1 and normal myofibroblast stromal cells WPMY-1. The relationship between the expression level of PCAT4 and the clinicopathological characteristics of patients with prostate cancer was analyzed. We detected the effects of PCAT4 on the cell proliferation and migration of prostate cancer cells by RNA interference, CCK-8 assay, and migration experiment. Results: The relative expression levels of PCAT4 in the prostate cancer tissues and adjacent tissues were 6.15±1.58 and 1.77±0.65, respectively （P<0.001）, while the relative expression levels of PCAT4 in the prostate cancer cell line PC3 and 22RV1 were higher than that in WPMY-1. The high expression of PCAT4 in the prostate cancer tissues was found in 30 cases of 48 cases, and 18 cases were low expression. The relative expression level of PCAT4 in prostate cancer tissues was associated with Gleason score and T stage （both P<0.05）, and was not associated with age, tumor size, lymph node metastasis and multiple lesions （all P>0.05）. In addition, the siRNA-mediated knockdown of PCAT4 in PC3 and 22RV1 cell lines significantly decreased the ability of proliferation and migration （all P<0.05）. Conclusion: The relative expression level of PCAT4 was elevated in prostate cancer. PCAT4 may promote the carcinogenesis and development of prostate cancer by affecting the proliferation and migration of prostate cancer cells, which can be used as a potential novel target for the diagnosis and treatment.

Abstract:Objective: The current study was aimed to mimic the physiology of human pulmonary vascular using an improved contact co-culture method containing human pulmonary artery endothelial cells(HPAECs) and human pulmonary artery smooth muscle cells (HPASMCs). Methods: HPAECs and HPASMCs were seeded on the opposite sides of a porous Transwell membrane that allowed formation of cell-cell contacts. The adhesion of HPAECs was determined by inverted phase contrast microscope and Hoechst staining. Flow cytometry was used to analyze the apoptosis of HPAECs and HPASMCs in different media and serum concentrations. The markers of PAECs and PASMCs were detected separately by immunofluorescence for identifying the characterization of the two kinds of cells. Results: The data from Hoechst staining showed that gelatin-precoated Transwell membrane facilitated the adhesion of HPAECs. Moreover, the apoptosis of HPAECs was increased under the hyperosmotic pressure. Compared with DMEM and RMPI 1640 medium, the endothelial cell medium(ECM) was more suitable for the contact co-culture system. Additionally, ECM with 1% endothelial cell growth supplement(ECGs) and 2% fetal bovine serum(FBS) promoted the growth of both HPAECs and HPASMCs. Moreover, co-culture adjustment showed no significant impact on characterization of PAECs and PASMCs in the aspect of cell morphologies and specific markers. Conclusion: ECM maintaining 1% ECGs and 2% FBS is better for setting HPAECS-HPASMCs contact co-culture system to mimic the physiology of human pulmonary vascular and to investigate the interactions between the two kinds of cells.

Abstract:Objective: To investigate the effect of environmental endocrine disruptor perfluorooctane suifonate(PFOS) on the cell damage and expression of inflammatory factors in mouse BV-2 microglial cells. Methods: The microglial cells were cultured by different concentrations of PFOS with various periods in vitro, and the activity of BV-2 cells was detected by using MTT method. Real-time PCR assay was performed to detect the mRNA expression of the inflammatory factors, including inducible nitric oxide synthesis(iNOS), tumor necrosis factor（TNF)-α and interleukin(IL)-6. At protein level, ELISA was used to analyze the excretion of IL-6 in BV-2 cells mediated by PFOS. Moreover, the signal pathway, especially the nuclear factor-kappa B(NF-κB), was evaluated by Western blot. Results: The BV-2 cells were incubated with varied doses of PFOS for 12 or 24 h, and then the cell viability was significantly decreased, which led to significant cell damage. ELISA data showed a significant incretion of the cytokine factor IL-6 after BV-2 cells were incubated with PFOS for 24 h. PFOS markedly up-regulated iNOS and IL-6 mRNA expression, while TNF-α was down-regulated. Therefore, we discovered that PFOS substantially activated and phosphorylated the NF-κB pathway in a dose dependent manner. In addition, with the increase of exposure time, the expression of p-NF-κB was also increased. Conclusion: Our results showed that PFOS markedly decreased the BV-2 cell viability, activated the NF-κB pathway, and excreted the cytokines. These results taken together might provide a new molecular mechanism supporting the PFOS-induced neural damage.

Abstract:Objective: To study on effects and mechanisms of nicotine on dopamine (DA) release in different brain regions of mice model with Parkinson’s disease(PD). Methods: PD mice were established by 1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine (MPTP). Motor function was detected by pole climbing test. The number of dopaminergic neuron in mice midbrain substantia nigra pars compacta(SNpc) region was detected using tyrosine hydroxylase immunohistochemistry. High performance liquid chromatography was used to determine the content of DA in different brain regions (the striatum， hippocampus and cortex). Striatal dopamine transporter (DAT) levels were detected by Western blot. Results: ①Nicotine significantly improved MPTP-induced motor function deficits in wild-type mice; ②Nicotine significantly prevented MPTP-induced dopaminergic neuron loss in wild-type mice SNpc; ③Nicotine significantly inhibited MPTP-induced decrease of striatal DA content in wildtype mice. Nicotine had no significant effect on MPTP-induced decrease of striatal DA content in α7-nicotinic acetylcholine receptors(α7-nAChRs) knockout mice. Nicotine had no obvious effect on DA content in the hippocampus and cortex of MPTP model in wild-type mice or knockout mice. ④Nicotine significantly increased the content of DAT in the striatum of MPTP model in wild-type mice. Nicotine had no effect on MPTP-induced decrease of DAT in the striatum of MPTP model in α7-nAChRs knockout mice. Conclusion: Nicotine inhibits the death of dopaminergic neurons in SNpc region of PD mice and improves striatal DAT levels through the activation of α7-nAChRs. Nicotine promotes the increase of striatal DA content， and exerts neuroprotective effects in PD mice.

Abstract:Objective：To investigate whether pyrroloquinoline quinone（PQQ） plays a treatment role in premature aging of skin in Bmi1-/- mice by inhibiting oxidative stress. Methods：We paired with littermates Bmi-1+/- between male and female mice，and selected the mice at the age of 7 weeks：wild type mice and normal diet （10 mice），Bmi1-/- mice and normal diet （10 mice），Bmi1-/- mice and add PQQ diet （10 mice）. HE staining，histochemistry，immunohistochemistry，and flow cytometry were peformed. Results：Compared with BKO mice，PQQ partially rescued total body phenotype，made hair of skin smooth，increased body weight and prolonged survival time. Compared skin thickness of PQQ+BKO mice ［（142.65±0.25）μm］ to BKO mice ［（78.45±0.35）μm］，the difference was statistically significant （P<0.01）. The percentage of total collagen positive area of skin in the PQQ+BKO group ［（25.64±0.28）%］ was significantly different from that in the BKO group ［（14.87±0.47）%,P<0.01］. The percentage of skin PCNA positive cells in the PQQ+BKO group ［（18.54±0.37）%］ was significantly different from that in the BKO group ［（5.85±0.64）%,P<0.01］. The positive area of skin fibrosis in the PQQ+BKO group ［（48.64±0.28）%］ was significantly different from that in the BKO group ［（87.64±0.46）%,P<0.01］. Compared with BKO mice （427.80±0.63），the levels of ROS in the skin of the PQQ+BKO group （298.17±0.25） were significantly decreased，and the difference was statistically significant（P<0.01）. Conclusion：The results of these studies indicated that PQQ played a role in protecting premature aging of skin induced by Bmi-1 defection through multiple aspects，such as promoting skin cell proliferation，reducing skin tissue fibrosis，scavenging ROS levels of oxygen free radical.

Abstract:Objective： To determine the short term efficacy and safety of different intensive anti-platelet treatments for patients who received percutaneous coronary intervention(PCI), but presented clopidogrel low response(CLR). Methods: The light transmittance aggregometer(LTA) was adopted to screen patients, who underwent PCI and presented CLR, and 150 clopidogrel low responders were consecutively recruited during their hospitalization. The included patients were randomized and assigned into three groups: ①the regular treatment group (Group A): taking aspirin 100 mg daily and clopidogrel 75 mg daily for one year; ②the intensive treatment group (Group B) : taking aspirin 100 mg daily and clopidogrel 150 mg daily for one month and then switched to regular anti-platelet treatment as group A for one year; ③the intensive treatment group (Group C): taking aspirin 100 mg daily and ticagrelor 90 mg twice daily for one month then turned to regular anti-platelet treatment as group A for one year. All recruited patients were followed up at one month after randomization, when the adenosine diphosphate induced platelet aggregation(PLADP) and arachidonic acid induced platelet aggregation(PLAA) was determined and clinical events were recorded. Results: There were no significant differences regarding the baseline clinical data and biochemical indexes among Groups A, B, and C, so were the baseline levels of PLADP（P=0.553）, and the baseline levels of PLAA（P=0.352）. At one month follow-up, the PLADP in Group B and Group C were both significantly lower than their baseline levels (P<0.001); and the PLADP level in Group C was significantly lower than that in Groups A and B; the PLAA levels of Groups A, B and C had no significant differences among the groups. The total incidences of in-stent thrombus, target vessel revascularization and cardiogenic hospitalization at 1 month were 20%, 10% and 4% in Group A, B and C, respectively(P=0.039). The incidences of minimal bleeding at 1 month were 10%, 14% and 8%, respectively in the three groups (P=0.044). The incidence of dyspnea in Group C was 6%, which was significantly higher compared to the other two groups(P=0.01). Conclusion: Double-dose clopidogrel or ticagrelor can effectively reduce the residual platelet aggregation for CLR patients; Ticagrelor can otherwise bring down the total incidences of in-stent thrombus, target vessel revascularization and cardiogenic hospitalization one month after the procedure, at the risk of increased dyspnea and minimal bleeding.

Abstract:Objective：To investigate whether preoperative serum soluble receptor activator of nuclear factor-кB ligand(sRANKL)，osteoprotegerin(OPG)，and sRANKL/OPG ratio are associated with the stabilization of restored sinus rhythm(SR) in persistent atrial fibrillation(AF) patients after valve surgery with concomitant modified maze procedure. Methods：We enrolled 114 patients with persistent AF who restored SR after valve surgery with concomitant modified maze procedure. According to the clinical follow-up outcomes at the third months after surgery，all the patients were divided into the AF recurrence group and the non recurrence group， respectively. Preoperative radial artery blood samples were obtained for measurement of serum sRANKL and OPG by ELISA. Results：Preoperative serum levels of sRANKL，OPG and sRANKL/OPG ratio were significantly higher in the AF recurrence group than those in the group with no AF recurrence. In multivariate Logistic regression analysis， except left atrial diameter，preoperative serum sRANKL level and sRANKL/OPG ratio independently predicted AF recurrence. According to ROC curve analysis，the best diagnostic values of serum sRANKL level and sRANKL/OPG ratio for predicting AF recurrence were 6.75 pmol/L and 0.87，respectively. The sensitivity，specificity, and accuracy of the recurrence predicted by sRANKL(＞6.75 pmol/L)were 89.7%，89.3% and 89.5%，respectively，and those by the sRANKL/OPG ratio(＞0.87)were 92.3%，98.7% and 92.1%，respectively. Conclusion：These findings suggest that preoperative serum sRANKL level and sRANKL/OPG ratio independently predict the recurrence of AF after valve surgery with concomitant modified maze procedure. Therefore，it can be used to screen patients who are suitable for the modified maze procedure or to be used as the target of early intervention to reduce the recurrence of AF.

Abstract:Objective： To investigate the difference of expression and prognostic significance of cyclin-dependent kinase 4 （CDK4） and Geminin in gliomas. Methods： Immunohistochemical was used to detect the expressions of CDK4 and Geminin in 70 human gliomas. Their relationships with clinicopathologic factors and survival time of patients were also analyzed. Results： The positive rates of CDK4 and Geminin in gliomas were 70.0%（49/70） and 75.7%（53/70），respectively. The positive rates of CDK4 in gradeⅡ， grade Ⅲ， grade Ⅳ of gliomas were 52.8%（21/36）, 84.6%（22/26） and 100.0%（8/8）, and the positive rates of Geminin in grad Ⅱ, Ⅲ and Ⅳ were 58.3%（21/36），92.3%（24/26） and 100.0%（8/8），respectively. A statistical significance of their expressions was observed among different grades （P<0.05）. The expressions of them were positively correlated. The positive expressions of Geminin and CDK4 were related to the tumor size and the number of nuclear division（P<0.05），and were not related to age，sex and necrosis of tumor tissue（P>0.05）. Kaplan-Meier univariate statistical analysis showed that the survival time of patients with positive CDK4 and Geminin expression group was significantly shorter than that of the negative CDK4 and Geminin expression group（P<0.001）. Conclusion： The expressions of CDK4 and Geminin are related to the grading of gliomas and prognosis. Combined detection of CDK4 and Geminin is helpful to judge the malignant degree and prognosis of gliomas.

Abstract:Objective: At present, there are few studies on the metastasis model of mediastinal lymph node (MLN) in stage I non-small-cell lung cancer (NSCLC). This study aimed to identify if patients with different clinical characteristics have different changes in metastatic rates between lobe-specific and non-specific MLN stations in patients with preoperative stage I NSCLC, so as to predict clinical indications of lobe-specific MLN dissection in patients with preoperative stage I NSCLC. Methods: We examined 609 patients with clinical stage I NSCLC for lobectomy with complete MLN dissection. We analyzed the metastatic rates of lobe-specific and other MLN stations with patient characteristics, including T stage, tumor histology, gender and age. Results: It was revealed that metastasis was limited to lobe-specific MLNs for stage Ⅰ patients meeting the following criteria: ①male, > 60 years, squamous cell tumor, and tumor≤5cm. ②male, ≤60 years, squamous cell tumor, and tumor≤ 3 cm. ③female,　squamous cell tumor, and tumor≤3 cm. ④adenocarcinoma and tumor≤2 cm. Conclusion: Lobe-specific mediastinal lymph node MLN dissection is suitable for the patients with characteristics mentioned above. For other patients, there was a considerable increasing risk of metastasis, and complete MLN dissection remains the most effective method.

Abstract:Objective： To compare the clinical effects between total hip arthroplasty (THA) and hemiarthroplasty (HA) for elderly femoral neck fracture. Methods: From 2007-2013, 385 elderly femoral fracture patients were treated by HA in the authors’ affiliated hospital. Among these patients, 82 patients (aged 80-85 years old) with the fracture type of Garden Ⅲ and Ⅳ were followed-up. According to these patients’ age, sex, height, weight and the classification of femoral neck fracture, we picked up 82 patients treated by THA for paired analysis. Preoperative Charlson comorbidity index(CCI), operation time, incision length, blood loss, complications, Harris hip score(HHS) of the last follow-up, hospital stay and expenses were recorded. Results: The mean follow-up time was 4.7 years. The difference of preoperative CCI between the two groups was statistically significant(P＜0.05). In the comparison of dominant blood loss, hidden blood loss, incision length, operative time, length of stay, and hospitalization costs, HA patients were better than THA patients (all P＜0.05). There were no significant differences in complication and HHS. Conclusion: HA can reduce blood loss, incision length, hospital stay and expenses, and is a good treatment for elderly patients with femoral neck fracture.

Abstract:Objective：To evaluate the root and canal morphology of mandibular anterior teeth by cone-beam computed tomography (CBCT). Methods: CBCT imaging data of 1 022 patients, who admitted in the Affiliated Stomatological Hospital of NJMU from June 2015 to June 2016, were inspected and divided into three groups according to tooth position, gender and age. The number of roots, root canals and root canal configuration were observed and analyzed statistically according to the method of Vertucci. Results: CBCT image data of 6 032 mandibular anterior teeth from 1 022 patients were obtained. All the mandibular central incisors and lateral incisors had a single root, while 0.9%(18/2 042) of canines had double. Multiple canals were found in 10.6%(211/1 990) of central incisor, 18.0%(360/2 000) of lateral incisors and 5.4%(111/2 042) of canines. The most prevalent canal type was Type Ⅲ. In multiple canal teeth, 89.9%(98/109) of central incisors, 87.3%(165/199) of lateral incisors and 48.0% (36/75) of canines occurred bilaterally. The incidence of multiple canals was 14.6%(57/397) of central incisors in the ＞30~40 years old group, 21.1% (62/294) of lateral incisor in the ＞40~50 years old group, and 8.4%(27/320) of canines in ＞50~60 years old group. The incidence of multiple root canals had statistical differences among the age groups. However, there were no statistical differences between females and males (11.15% vs. 11.53%, P>0.05). Conclusion: We should pay more attention to the occurrence of the multiple canals in mandibular anterior teeth for root canal treatment. The incidence of the multiple root canals has a certain correlation with age.

Abstract:Objective：To analyze current situation of human immunodeficiency virus(HIV)-infected injecting drug users after 10 years of the implementation of methadone maintenance treatment(MMT). Methods: Data of HIV-infected cases and patients who received MMT were downloaded from HIV/AIDS reporting system to analyze current situation of MMT for HIV-infected cases. Results: A total of 430 HIV infected cases reported by medical establishments in Jiangsu had never got service from MMT clinics. One hundred and ninety HIV infected cases had been accepted by MMT clinics, and 31.58% of them persisted by 2015. Compared with those outside MMT clinics, HIV infected cases in MMT were older(P<0.001), with longer HIV antibody positive status (P<0.001), higher proportion of Han ethnicity(P<0.001), and higher education level(P<0.001). Conclusion: Outpatient coverage of MMT should be enlarged, so that more HIV-infected drug users could get convenient service. Comprehensive intervention measures are recommended to improve the service quality of MMT clinics.

Abstract:Objective: To evaluate the effectiveness of intrathecal injection of methotrexate (MTX) and dexamethasone (DXM) combined with glucocorticoid in treatment for neuropsychiatric systemic lupus erythematosus(NPSLE). Methods: The clinical literatures about intrathecal injection of MTX and DXM combined with glucocorticoid in treatment of NPSLE were collected from January 1996 to December 2016. Meta-analysis was performed by comparing control with treatment group. The control group was treated with glucocorticoid or glucocorticoid combined with cyclophosphamide. The treatment group was treated with intrathecal injection of MTX and DXM combined with glucocorticoid. Results: A total of 6 clinical trials with 256 NPSLE patients were included. There were 126 patients in the control group and 130 patients in the treatment group. Meta-analysis indicated that the treatment group was superior to control group in cerebrospinal fluid pressure, cerebrospinal fluid protein, erythrocyte sedimentation rate, and systemic lupus erythematosus disease activity index (SLEDAI) (all P<0.01). Conclusion: The curative effect of treatment group is superior to that of the control group. However, due to the low methodological quality of the literature, it still needs large sample, multi-center, high-quality clinical research trails.