Abstract:Objective：This study aims to investigate the effect of Alamandine，a peptide derived from the decarbonization of angiotensin（1-7），on the adipogenic differentiation of adipose-derived mesenchymal stem cell（ADSC）in rats，and the mechanism of Alamandine regulating lipogenic differentiation. Methods：Rat ADSCs were treated with Alamandine at different concentrations（0.1 μmol/L，1.0 μmo/L，10.0 μmol/L），lipid droplets were observed by light microscopy，intracellular triglyceride and total cholesterol were determined，the degree of lipid differentiation was detected by oil red O staining，and the protein associated with adipogenic differentiation was detected by Western blot. After determining the optimal concentration，10 μmol/L Alamandine was used to detect the adipogenic differentiation degree of rat ADSCs in different days. Angiotensin Ⅱ（Ang Ⅱ）and Alamandine dealt with rats ADSCs，same method to detect a concomitant with differentiation，detect the effect of Ang Ⅱ and Alamandine on adipogenic differentiation. Rat ADSCs were treated with Mas associated G protein coupled receptor D（MrgD）antagonist D-pro7，and adipogenic differentiation was detected by the same method，so as to further verify the role of Alamandine in promoting adipogenic differentiation. Results：Alamandine treatment of rat ADSCs with different concentrations showed that Alamandine promoted adipogenic differentiation of rat ADSCs in an obvious dose-dependent manner. ADSCs of rats treated with 10 μmol/L Alamandine were detected at 1，3，6 and 10 days，respectively. With the increase of treatment days，the degree of adipogenic differentiation gradually increased and reached its peak on the 10 th day. Ang Ⅱ single processing rat ADSCs inhibited adipogentic differentiation and the effect of Alamandine contributing to fat differentiation is weakened with Ang Ⅱ. MrgD antagonist D- Pro7 also inhibited the lipid differentiation effect of Alamandine on rat ADSCs. Conclusion：Alamandine promotes lipid differentiation through MrgD on rat ADSCs. This may provide a new way to treat obesity.

Abstract:Objective：This study aims to investigate the effect of C5a on the proliferation and migration of non-small cell lung cancer（NSCLC）cells and its potential molecular mechanism. Methods：The C5a receptor（C5aR）expression in the human normal bronchial epithelial cell line BEAS-2B and three NSCLC cell lines（H1703，PC9，H1299） was examined by RT-PCR and Western blot（WB）analysis. PC9 cell were stimulated with various concentrations of C5a，and cell proliferation and migration were examined by CCK-8 and scratch test，respectively. The PC9 cells were treated with C5a，and then the expression and phosphorylation of Akt1，ERK1/2 and PKC-α were examined by WB. PC9 cells were treated with Akt1 inhibitor（Perifosine）and ERK1/2 inhibitor（U0126），respectively，followed by C5a stimulation，and then WB was used to examine the expression and phosphorylation levels of Akt1 and ERK1/2 and their upstream and downstream regulatory relationships. The effects of Perifosine and U0126 on PC9 cell proliferation and migration were determined by CCK-8 and scratch test，respectively. Results：The expression level of C5aR in PC9 cells was obviously higher than that of other cells. C5a could significantly promote the proliferation and migration of PC9 cells. In addition，the phosphorylation levels of both Akt1 and ERK1/2 were markedly enhanced in the PC9 cells induced by C5a，but the protein expression did not show significant change. Furthermore，Akt1 and ERK1/2 inhibitors markedly down-regulated the proliferation and migration of PC9 cells caused by C5a stimulation. Akt1 inhibitors not only attenuated Akt1 phosphorylation，but also attenuated ERK1/2 phosphorylation. ERK1/2 inhibitors only attenuated its own phosphorylation. Conclusion：C5a induces the proliferation and migration of NSCLC cells through activation of Akt1/ERK1/2 pathway.

Abstract:Objective：This study aims to explore the role and potential molecular mechanism of microRNA（miR）-190 in aging-related energy metabolism disorders. Methods：Real-time fluorescent quantitative PCR（RT-PCR）was used to detect the expression level of miR-190 in the adipose tissue of aging mice；a cellular senescence model was constructed and RT-PCR was used to detect the changes of miR-190 in adipocytes；RT-PCR was used to detect the changes in thermogenesis genes in the adipocytes transfected with miR-190；the target genes of miR-190 were verified by the dual luciferase reporter gene experiment；the metabolic homeostasis was determined in aged mice injected with miR-190 antagomir. Results：During the aging process，the expression of miR-190 in adipose tissue was elevated；in the senescent cell model，the expression of miR-190 was also up-regulated；overexpression of miR-190 in adipocytes would lead to a decrease in the expression of thermogenesis genes；the key genes for thermogenesis PRDM16 is the direct target gene of miR-190；inhibiting the level of miR-190 in aging mice can help improve the energy metabolism homeostasis of aged mice. Conclusion：miR-190 may affect adipose tissue heat production by targeting PRDM16，which in turn affects energy metabolism homeostasis and accelerates aging.

Abstract:Objective：This study aims to explore the role of deubiquitinating enzyme YOD1 in liver nutrient metabolism. Methods：The expression of YOD1 in the liver of HFD-fed C57BL/6 mice，db/db mice and in Hepa1-6 cells after oleic acid（OA） treatment was checked by Q-PCR. Then its tissue distribution in C57BL/6 mice and its expression under different nutritional conditions was detected by Q-PCR. The content of triglycerides in Hepa1-6 cells treated with OA was determined by TAG enzymetic kit，and lipid droplets were observed by Oil Red O staining. Moreover，the mRNA and protein levels of related genes in cells were detected by Q-PCR and Western blot. Results：After a short-term HFD-fed，the mRNA level of YOD1 in the liver decreased significantly. But compared with that of db/+ mice in control group，the expression of YOD1 was no change in db/db mice.However，its expression reduced obviously in OA-treated Hepa1-6 cells. Besides，YOD1 was mainly distributed in the liver，and we observed that fasting led to increased mRNA level of YOD1 in mice liver while YOD1 decreased notably after refeeding. Further，YOD1 overexpression remarkably ameliorated OA-induced steatosis in Hepa1-6 cells by inhibiting SREBP-1c cleavage.　Conclusion：YOD1 expression is regulated by nutritional status and modulates lipid metabolism in hepatocytes by inhibiting SREBP-1c cleavage.

Abstract:Objective：This study aims to investigate the expression and function of long non-coding RNA ENSMUST00000127391（lncRNA 127391）in mice mesangial cell（MMC）cultured with high glucose. Methods：The recombinant plasmid pCDH-lncRNA 127391 was successfully constructed in our group. In this study，the recombinant plasmid pCDH-lncRNA 127391 and control plasmid pCDH were transfected into MMC cells respectively，and the expression of lncRNA 127391 was detected by RT-qPCR. The fibrosis markers of fibronectin（FN） and type Ⅰ collagen（Col-1） and proliferation markers of Cyclin D1 and proliferating cell nuclear antigen（PCNA） were detected by Western blot. CCK8 assay was used to detect the proliferation of MMC. Flow cytometry was used to study the effect of lncRNA 127391 on the apoptosis of MMC. Results：Overexpression of lncRNA 127391 in MMC cells was successfully constructed. CCK8 showed that the overexpression of lncRNA 127391 decreased the proliferation rate of MMC cells. And the expression of Cyclin D1，proliferating cell nuclear antigen PCNA，FN and Col-1 in MMC cells decreased after overexpression of lncRNA 127391. Flow cytometry showed that the overexpression of lncRNA127391 increased the number of apoptotic MMC cells. The differences were statistically significant（P < 0.05）. Conclusion：lncRNA 127391 can inhibit the proliferation and fibrosis of MMC cells cultured with high glucose.

Abstract:Objective：Bioinformatics analysis was applied to explore the differentially expressed genes（DEG）in normal women with high breast density and low breast density and analyze their enrichment pathways，so as to provide theoretical basis for early diagnosis，targeted therapy and prognosis assessment of breast cancer patients with dense breast. Methods：GSE38506 data set was obtained from the GEO database and DEGs were screened using GEO2R. Enrichment analysis of GO and KEGG was performed using DAVID database. CytoHubba plug-in of STRING and Cytoscape software was used for protein interaction network analysis and Hub gene screening. Finally，the online tool bcGenExMiner was used to analyze the prognosis of these 5 Hub genes. Results：A total of 830 significantly DEGs were screened from GSE38506 gene chip，among which 442 genes were up-regulated and 388 genes were down-regulated. Enrichment analysis showed that DEGs were mainly involved in signal transduction，positive regulation of GTPase activity，innate immune response，cell adhesion，cytoplasmic membrane，cell surface，cell connection，ATP binding，actin binding，transcription factor binding，and so on. A total of 2 core modules and 5 Hub genes were screened，including EGFR，JUN proto-Oncogene，CDC42，SRC proto-oncogene，RAC1，and they were all associated with breast cancer prognosis. Conclusion：In this study，5 Hub genes related to mammographic density in healthy women were screened through bioinformatics，which may have certain influence on the occurrence，development and prognosis of breast cancer，and are potential molecular markers and new targeted therapy sites for breast cancer patients with dense breast.

Abstract:Objective：This study aims to establish a stable cell line with high expression of anti-plague F1 chimeric antibody，express and purify the chimeric anti-plague F1 antibody and identify its activity. Methods：Total RNA of murine hybridoma cell line 4C6 was extracted and reverse transcribed into cDNA. The variable region genes of light chain and heavy chain of anti-F1 antibody were obtained by PCR and the gene synthesis chimeric antibody heavy chain and light chain gene，and cloning in eukaryotic expression vector pMH3 plasmid of transfection cells. CHO - S stable expression is obtained by G418 screening F1 chimeric antibody cell lines. Supernatant was collected and purified after suspension culture. The specific binding ability with F1 antigen was detected by ELISA. The binding ability of chimeric antibody and mouse parent antibody to antigen was compared by competitive ELISA，the antibody affinity was determined，and the antibody properties were analyzed by mass spectrometry. Results：Stable cell lines secreting chimeric antibody against plague antigen F1 were successfully obtained. The antibody could specifically bind to F1 antigen，and the affinity of the antibody was 2.303×10-9 mol/L. Conclusion：In this study，chimeric antibody against plague F1 was successfully prepared，which laid a foundation for subsequent animal experiments and clinical drug development.

Abstract:Objective：This study aims to construct a Y chromosome haplogroup single nucleotide Polymorphism multiplex system（Y-SNP system）and evaluate its application value in familial searching of forensic genetic. Methods：The Y-SNPs that widely distributed in East Asia were screened. The system was constructed based on capillary electrophoresis（CE）genotyping platform，and its performance and accuracy were evaluated and verified. Twenty male samples from four pedigrees were collected. And the genotypes of Y-SNP and Y chromosome short tandem repeat（Y-STR）genetic markers were performed using the Y-SNP system and DNA TyperTM Y29 kit respectively. The differences of haplogroups and haplotypes in each pedigree were analyzed，and the network was used to visualize the mutation. Results：A total of 74 Y-SNPs were selected and constructed in double-tube of multiplex system. The SNP genotypes obtained by Y-SNP system were completely consistent with Sanger sequencing results. The low DNA detection limit was 0.14 ng. In addition，this system was suitable for blood stain，oral swabs，nails，hair root. Except chimpanzees and rhesus monkeys，there was no specific peak in non-human DNA samples. In P1 pedigree，the Y-SNP haplogroups and Y-STR haplotypes of samples were identical. In P2 pedigree，the Y-SNP haplogroups of all samples were identical and Y-STR haplotypes were different（mismatched loci ≤5）. In P3 pedigree，the Y-SNP haplogroups of all samples were different and Y-STR haplotypes were different（mismatched loci >5）. In P4 pedigree，the Y-SNP haplogroups of all samples were identical and Y-STR haplotypes were different（mismatched loci >5）. Conclusion：The combination of Y-SNP and Y-STR can improve the accuracy of familial searching.

Abstract:Objective：This study aims to explore the possible targets of cognitive impairment in children with childhood absence epilepsy（CAE） by analyzing the changes of white matter fiber integrity in children with CAE. Methods：From November 2011 to November 2020，there were 29 children with CAE，11 males and 18 females，with an average age of 8.4±1.9 years，from Nanjing Children’s Hospital or Nanjing Brain Hospital；and 24 healthy children were matched in age and sex，9 males and 15 females，with an average age of 7.9±1.4 years old. The DTI and T1 data of all subjects were collected by Siemens 3.0T MRI. Twenty white matter fiber bundles were extracted by AFQ，and the fractional anisotropy（FA） and mean diffusivity（MD） values on 100 equal sub-nodes of each fiber bundle were extracted. The differences between CAE group and control group in each node were compared. The statistical methods were independent sample t-test and multiple comparison correction. Result：The FA decreased and MD increased in most fiber bundle nodes of CAE children，especially in uncinate and arcuate. Conclusion：The study suggests that there is a change of white matter fiber bundle integrity in children with CAE，especially in uncinate and arcuate. The damage of uncinate and arcuate is the main targets of cognitive impairment in CAE.

Abstract:Objective：This study aims to investigate the relationship between middle cerebral artery（MCA）plaque features on high-resolution vessel wall imaging（HR-VWI）and acute ischemic stroke/TIA recurrence. Methods：The HR-VWI and clinical data of 105 patients with atherosclerotic plaques on MCA were retrospectively analyzed. They were divided into group 1，with the first episode of acute ischemic stroke/TIA（n=61）；group 2，with recurrent acute ischemic stroke/TIA（n=19）；group 3，with no occurrence of clinical event（n=25）. The degree of stenosis，remodeling index and plaque features（enhancement ratio，enhancement grade，plaque burden，T1 signal intensity，and plaque distribution）were measured and compared among groups using analysis of variance，chi-square，Fisher’s exact test or Kruskal-Wallis test. Regression models investigated risk factors for recurrent stroke/TIA. Results：Plaque burden，enhancement ratio，remodeling index were all significantly higher in group 1 and 2，compared to group 3（all P < 0.05）. Higher plaque burden（P=0.005），higher stenosis degree（P=0.006）and more concentric plaque（P=0.008） were found in group 2 compared to group 1（P < 0.05）. Multivariable logistic regression shows plaque burden was the only significant plaque marker associated with recurrent stroke/TIA（OR=1.075，95% CI 1.019~1.133，P=0.008）. Conclusion：Higher plaque burden of MCA was independently associated with recurrent acute ischemic stroke/TIA and can be an effective imaging marker for alarming the ischemic stroke recurrence.

Abstract:Objective：This study aims to characterize the gene mutation in myeloid malignancies with normal karyotype by exploring multiple gene mutations. Methods：Tatal 102 acute myelogenous leukemia（AML） with normal karyotype and myelodysplastic syndrome（MDS）paticnts were retrospectively analyzed in Changzhou Second Affiliated Hospital of Nanjing Medical Univcrsity and the First Affiliated Hospital of Suzhou University. Targeted second generation sequencing were performed using a custom-designed 49-gene panel. CALR，FLT3 internal tandem duplication（FLT3-ITD），NPM1 and CEBPA mutation were detected by Sanger sequencing. Results：①Together，gene mutations accounted for a considerable frequency of 98.8% AML patients. Coexistence of ≥ 3 mutations was identified in 52.4% patients. The most commonly mutated gene was NPM1（35.4%），followed by FLT3-ITD（25.6%），CEBPA double mutations（24.4%），DNMT3A（19.5%），TET2（18.3%），NRAS（13.4%），RUNX1（11.0%）and CSF3R（11.0%）mutations. ②The gene mutations were present in 90% MDS patients. Coexistence of ≥ 3 mutations was in 55.0% patients. The most commonly mutated gene was RUNX1（35.0%），followed by ASXL1（25.0%），SF3B1（15.0%），FLT3-TKD（15.0%）and BCOR（15.0%）mutations. ③The incidence of mutation was similar in AML patients and MDS patients（P=0.097）. The mutation rate of each of NPM1 and CEBPA double mutations was higher in patients with AML（P < 0.05），while RUNX1，ASXL，SF3B1 and BCOR mutations were identified more frequently in MDS patients，both differences were significant（P < 0.05）. ④Gene aberrations involved in DNA methylation（DNMT3A，TET2，IDH1/2）and receptors/kinases（FLT3-ITD，FLT3-TKD，JAK1，JAK2，JAK3，c-KIT，PDGFRA，PDGFRB，MPL，CSF3R，NOTCH1，IL7R）significantly predominated in AML while post-translational chromatin modification（EZH2，ASXL1/2，SETD2）and RNA splicing（SRSF2，SF3B1，ZRSR2，U2AF1）significantly predominated in MDS（P < 0.05）. Conclusion：Different functional mutation combinations revealed multiple sub-clones in myeloid malignancies with normal karyotype. The genomic landscape of AML patients was different from MDS patients.

Abstract:Objective：This study aims to study the effect of elevated hs-cTnT on clinical outcome and mortality in AIS patients who were under intravenous thrombolytic therapy after 90 days and explore the factors affecting hs-cTnT elevation. Methods：The clinical data of 262 patients were collected and retrospectively analyzed. They were divided into hs-cTnT elevation group（n=70）and hs-cTnT normal group（n=192），poor outcome group（n=94）and good outcome group（n=168），mortality group（n=24）and survival group（n=238）. Groups comparison，univariate regression analysis，multivariate regression analysis were performed. Results：Multivariate regression analysis results showed that，factors associated with hs-cTnT elevation were elderly（OR=1.062，95%CI：1.029~1.097，P < 0.001），male patients（OR=4.35，95%CI：1.982~9.545，P < 0.001）and admission NIHSS score（OR=1.062，95%CI：1.019~1.106，P=0.004）. Factors associated with poor outcome in AIS patients who were under intravenous thrombolytic therapy after 90 d were elderly（OR=1.031，95%CI：1.003~1.059，P=0.028）and admission NIHSS score（OR=1.086，95%CI：1.042~1.131，P < 0.001）. Factors associated with mortality in AIS patients who were under intravenous thrombolytic therapy after 90 d were hs-cTnT elevation（OR=5.31，95%CI：1.025~27.517，P=0.047），admission NIHSS score（OR=1.126，95%CI：1.057~1.200，P < 0.001）and high blood pressure（OR=4.254，95%CI：1.387~13.046，P=0.011）. Conclusion：Elderly，male patients in AIS with high NIHSS scores at admission had higher levels of hs-cTnT. Thus hs-cTnT could be used as a possible marker to predict 90 d mortality after intravenous thrombolysis in AIS patients.

Abstract:Objective：This study aims to analyze the clinical features of patients with sepsis secondary to liver abscess and find the early diagnostic indicators. Methods：A total of 237 patients with liver abscess diagnosed and treated in the First Affiliated Hospital of Nanjing Medical University from January 2017 to April 2021 were selected as the research subjects. They were divided into two groups：the non-sepsis group and the sepsis group，and the clinical characteristics of the two groups were analyzed. The data was analyzed using SPSS 22.0. Results：Diabetic patients were more prone to liver abscess（72.6%）. Patients in the sepsis group were retrospectively evaluated according to qSOFA. Compared with the non-sepsis group，the differences in respiratory rate ≥22 cpm（0.007），GCS<13（P < 0.001）and systolic blood pressure were statistically significant. The white blood cell count of patients with sepsis group［（13.81±6.89）×109/L vs. （10.91±6.89）×109/L，P < 0.001］，the neutrophil ratio［（84.29±8.15）% vs. （79.86±12.68）%，P=0.021］，C-reactive protein（P=0.048）and procalcitonin（P < 0.001）were higher than those in the non-sepsis group. Blood glucose control was worse in the sepsis group（P=0.016）. There was no difference in abscess growth location and bacteriological culture between the two groups. The leading cause of liver abscess was Klebsiella pneumoniae（76.8%）. Conclusion：Diabetic patients are prone to secondary liver abscess. The qSOFA can be used for the early judgment of the severity of liverabscess. WBC count，neutrophil ratio，C-reactive protein and procalcitonin can be used for early diagnosis of liver abscess and further prediction of disease development. Klebsiella pneumoniae is the main pathogen of liver abscess.

Abstract:Objective：This study aims to analyze the risk factors of intracranial hemorrhage（IH） in patients with acute ischemic stroke（AIS） treated with intra-arterial thrombectomy（IAT）in extended time window，and to establish the predictive model for guiding clinical decision. Methods：From January 2018 and June 2020，clinical data of 146 AIS patients treated with IAT in extended time window were retrospectively analyzed. Patients were divided into hemorrhage and no-hemorrhage group. Demographic，clinical and imaging data were compared between two groups. Logistic regression（LR）analysis was applied to clarify the risk factor of IH after IAT，and to establish the predictive model. Receiver operator characteristic curve（ROC）analysis was used to evaluate the performance of the model for predicting IH after IAT. Results：IH occurred in 48（32.9%）patients. Compared with no-hemorrhage group，hemorrhage group showed higher baseline National Institute of Health stroke scale（NIHSS）（P < 0.001），higher numbers of stent thrombectomy（P=0.049）and lower baseline alberta stroke program early computed tomography score（ASPECTS）（P < 0.001）. LR analysis indicated that，high NIHSS（P=0.001）and low ASPECTS（P < 0.001）were risk factors of IH. The predictive model was as follows：hemorrhage risk value =-0.535+0.130×NIHSS-0.597×ASPECTS. The predictive model showed an area under the ROC curve，sensitivity and specificity of 0.875，0.854 and 0.837，respectively. Conclusion：In the AIS patients in extended time window，the patients with high baseline NIHSS and low ASPECTS were prone to IH after IAT. The predictive model can provide reference for prevention of IH and clinical observation.

Abstract:Vascular anomalies are classified as vascular tumors and vascular malformation，common in childhood. The mammalian target of rapamycin inhibitor sirolimus has proved to be efficacious for many vascular anomalies by inhibiting cell proliferation and angiogenesis. Based on the latest International Society for Study of Vascular Anomalies （ISSVA） classification system，it was described the application of Sirolimus of vascular anomalies in children in this article. The purpose of this paper is to facilitate sirolimus precision dosing in children with vascular anomalies.

Abstract:Cardiovascular disease is a serious threat to human health，and it is also the main cause of death in the world. The traditional two dimensional（2D）cell model and animal model have their own limitations，which cannot be fully applied to the mechanism and treatment of cardiovascular disease. Organoid is a kind of three dimensional（3D）culture in vitro and simulates the cell structure of organ in vivo，which can more accurately retain the biological characteristics and functions of cells in vivo. It is a new direction and idea for the research of cardiovascular disease. Now，many studies have reported the construction methods of cardiac organs and their applications in drug experiments，disease models，regenerative medicine in treatment and so on. In this review，it was summarized the construction methods of cardiac organs and their clinical applications，and also put forward their development prospects and limitations.

Abstract:Chronic obstructive pulmonary disease（COPD）is a common chronic lung disease，and the related pathogenesis has not been fully clarified. In recent years，the role of respiratory tract and gut microbiota in the pathogenesis and treatment of COPD has been attracted extensive attention worldwide. This article reviews related literatures on the distribution and abundance of respiratory tract microbiota in COPD patients，and the influence of gut microbiota on the pathogenesis of COPD，in order to expand the understanding of the pathogenesis of COPD and the influence of intestinal-pulmonary axis on the pathogenesis of COPD，and provide ideas for exploring new prevention and treatment strategies for COPD.

Abstract:Glaucoma is a common neurodegenerative disease with complex pathogenesis，which seriously threatens human eye health. The establishment of glaucoma animal model provides a research basis for the occurrence，development and intervention of glaucoma. Based on the brief introduction of glaucoma，this paper describes the current construction methods of animal models of glaucoma from the two perspectives of spontaneous and artificially induced glaucoma animal models. An animal model of glaucoma can only simulate a certain type of glaucoma or one aspect of the pathological changes of glaucoma. Therefore，we should choose a reasonable animal model according to different experimental needs. With the development of biotechnology and basic scientific research，as well as the in-depth understanding of the pathogenesis of glaucoma，it is expected to further improve the existing models or develop new induction mechanisms to overcome the current limitations，so that we can better understand and intervene the occurrence and development of glaucoma in the future.

Abstract:The disadvantages of traditional therapy for peri-implantitis have gradually shown. Er：YAG laser can be used to make up for the deficiency of traditional therapy for the functions of sterilization，disinfection，hemostasis and photobiological regulation. However，how to decontaminate the implant without damaging the surrounding tissue？How to decontaminate the implant without damaging the surface characteristics and biocompatibility of itself？These have always been a concern of researchers. In addition，more and more attention has been paid to the concrete effect of Er：YAG laser on the therapy of peri-implantitis. We will discuss the above issues in this review.