Objective To investigate the bionomics of mesenchymal stem cells (MSCs) derived from Wharton's jelly of human umbilical cord. Methods Umbilical arteries, vein and umbilical cord tunica externa were removed, and the remaining tissue (Wharton's jelly) was harvested. To gather MSCs, the umbilical cord was cut into small fragments and digested with 0.075% type Ⅰ collagenase, or the small fragments were cultured with DMEM. The cells derived from Wharton's jelly of umbilical cord were serially subcultivated, the growth curve was drawn, and the identification of living cells was made for studying the cell growth kinetics. The surface antigens were detected with flow cytometry, the cartilage markers were detected by histochemistry and immunohistochemistry, and the expressions of Sox-9 and Col-2A1 mRNA were analyzed by RT-PCR. Results The confluence time of primary culture cells was 3-5d in cells harvested with enzyme digestion, and 10-15d in cells harvested with micro mass. The results of flow cytometry showed that the MSCs derived from umbilical cord expressed CD44 and CD105; the expression of HLA-ABC was positive, while HLA-DPDQDR was negative. There was no significant change on the immunophenotype of umbilical cord MSCs after cryopreservation and thawing. The findings of histochemistry and immunohistochemistry showed that the expression of chondrocyte markers in Wharton's jelly MSCs was weakly positive. The results of RT-PCR showed the chondrocyte markers Sox-9 and Col-2A1 were positively expressed in Wharton's jelly MSCs. Conclusion The MSCs derived from Wharton jelly of human umbilical cord do not differentiate into hematopoietic cells, may express the chondrocyte markers (they are suggested to have characteristics of pre-chondrocytes), and is expected to be a new type of stem cells of tissue engineering cartilage.

To study the effect of sphingosine-1-phosphate (S1P) on the cardiomyogenic differentiation of human umbilical cord mesenchymal stem cells (UC-MSCs) and human adipose-derived mesenchymal stem cells (AD-MSCs), we seeded the cells in the culture plates and used cardiomyocyte culture medium (CMCM) combining with different concentration of S1P to induce UC-MSCs and AD-MSCs in vitro for 7, 14 and 28 days. Cardiomyogenic differentiations were identified through immunofluorescence staining, and the results were observed with fluorescence microscopy and confocal microscopy. The effects of S1P and CMCM on cell activity were evaluated by the methyl thiazolyl tetrazolium assay. The functional characteristic similar to cardiomyocytes was evaluated through detecting calcium transient. Our results showed that cardiomyogenic differentiation of UC-MSCs or AD-MSCs were enhanced with S1P concentration increasing, but cell activities declined. Results showed that the suitable differentiation time was 14 days, and the optimal concentration of S1P was 0.5 micromol/L. When working together with CMCM, S1P could promote the differentiation of UC-MSCs or AD-MSCs into functional cardiomyocytes, giving rise to specific electrophysiological properties (the calcium transient). Taken together, our results suggested that S1P could promote the differentiation of UC-MSCs or AD-MSCs into functional cardiomyocytes when being cultured in CMCM.
Adipose Tissue ; cytology ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Culture Media ; Humans ; Lysophospholipids ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Myocytes, Cardiac ; cytology ; Sphingosine ; analogs & derivatives ; pharmacology ; Umbilical Cord ; cytology

Objective To explore the HLA-A2 restriction and immunogenicity of 5 previously identified HCV-speeific CTL epitopes. Methods Based on T2 cell, to explore the HLA-A2 restriction of previously identified HCV-specific CTL epitopes by MHC-peptide complex stabilization assay;To detect pep-tide-specific CTL in HLA-A2~+ PBMC stimulated by HLA-A2-restricted peptides by intracellular cytokine staining(ICS) and ELISPOT; To explore the cytotoxicity of peptide-specific CTL to same peptide-loaded T2 cells (target cells) by CTL cytotoxicity test. Results Among 5 previously identified CTL epitopes NS4b_78 (SMMAFSAAL) and NS5a_367 (TVSSALAEL) have high-affinity for HLA-A2 molecules(FI 1) ;ELISPOT results shown that NS4b_78(SMMAFSAAL) and NSSa_367(TVSSALAEL) induced high levels of IFN-γ-se-creting cells [(60±6) SFC/10~4 PBMC vs (4±1 ) SFC/10~4 PBMC, P < 0.01 ; (10 ± 3 ) SFC/10~4 PBMC vs (2±1 ) SFC/10~4 PBMC, P <0.01, respectively] ;ICS results indicated that there were high percentages of CD8~+ IFN-γ~+ T cells in total CD8~+T cells stimulated by these peptides [(2.33 ±0.22 ) % vs (0.05±0.01)%, P <0.001 ; (0.36±0.06)% vs (0.03±0.01)%, P <0.001, respectively]. Furthermore,peptide-specific CTL could effectively kill same peptide-loadcd T2 cells. Conclusion NS4b_78 (SMMAF-SAAL) and NSSa_367 (TVSSALAEL) were identified as HLA-A2-restricted CTL epitopes which could in-duce immune response in vitro.

A 21-year-old male was hospitalized for recurrent swelling of lower limbs (lymphedema) for 1 year and worsening of it for 1 week.Physical examination revealed several smooth,firm enlarged lymph nodes of the neck,groin without apparent tenderness measuring about 1 cm in diameter.Cardiac and pulmonary auscultation showed no obvious abnormality.The abdomen was soft on palpation without tenderness or rebound tenderness.Skin examination revealed swelling of both lower limbs,especially the left lower limb,as well as scattered irregularly sized,dark erythematous patches with a wood-like consistency on the swollen lower limbs,with high temperature but no tenderness.Elevated peripheral eosinophil count was observed before and after admission,with the eosinophil percentage higher than 70％.Vascular ultrasonography showed thrombosis in the right anterior tibial artery,left dorsal artery of foot and lower portion of the left great saphenous vein.Multiple enlarged lymph nodes were found by computed tomography in the mediastinal,bilateral axillary,retroperitoneal regions and around the abdominal aorta.Lymph node and bone marrow biopsies showed eosinophilia.Histopathology of lesions on the extensor aspect of the left medial thigh and left lateral malleolus showed massive eosinophilic infiltration and lymphatic dilation in the dermis,as well as eosinophil emboli in some lumens.The FIP1L1-PDGFRA fusion gene was undetected.A diagnosis of idiopathic hypereosinophilic syndrome was finally made.The symptoms rapidly regressed after glucocorticoid treatment.

Objective To probe the immunological traits of mesenchymal stem cells derived from umbilical cord Wharton's jelly (WJMSCs). Methods The diced Wharton's jelly which was from healthy fullterm birth human umbilical cord was cultured. The mesenchymal stem cells were identified with mesenchymal stem cells markers expression by flow cytometry and multiple differentiation ability. The expression of MHC- Ⅰ / Ⅱ, costimulatory molecules (CD40, CD80 and CD86) was detected with flow cytomctry, immunocytochemistry, and RT-PCR. The expression of immune inhibitors like HLA-G, IDO, and PGE2 was detected by immunocytochemistry and RT-PCR. The expression of immune-related molecules as IL-10, TGF-β, FGF and VEGF was detected with antibody microarray and western blot. Further more, to clarify the in vivo immune reaction of hWJMSCs, we fabricated the hWJMSC-scaffold constructs and implanted them into the rabbit backs. The lymphocyte infiltration and implanted cell survival observed with immunofluorescence. Results After culturinge of diced Wharton's jelly tissue, we obtained spindle-shaped cells. With differentiation medium, the cells can differentiate into osteoblasts, chongdrocytes, adipose cells and schwann cells. Expression of MHC, costimulatory molecules, and a series of immune suppressive-related molecules was found. Immune inhibitors as HLA-G, 1DO, PGE2, and immune suppressive related molecules as HGF, VEGF, TGFand IL-10 were positively expressed. But the cells did not express MHC-Ⅱ. No immune rejection was observed in vivo after implantation of hWJMSC-scaffold constructs. Conclusion It can be concluded that hWJMSCs have very low immunogenicity, which means the cells have potential to induce immune tolerance.The hWJMSCs do not provoke immune rejection in vivo.

Objective: To investigate the clinicopathological features and differential diagnosis of adamantinoma of long bone. Methods: Seven cases of adamantinoma on long bone were selected at Jiangsu Province People′s Hospital from June 2012 to May 2018. Clinicopathologic details, immunohistochemical and molecular analysis were performed,and the relevant literature reviewed. Results: There were 6 males and 1 female patients,age ranging from 21 to 60 years (mean 38 years). Six cases were on the right side and one case was on the left; in five cases the tumors arose from tibia, one from patella and one from humerus. Microscopically,tumour cells were mainly composed of spindle cells arranged in bundles or braids,with irregular epithelial island. Immunohistochemically,the epithelial island expressed high molecular weight cytokeratin but not CK8/18. Both epithelial and spindle components expressed vimentin. One case that was microscopically similar to intraosseous synovial sarcoma did not show SYT gene rearrangement. Clinical follow-up was available for five patients: one patient had axillary metastases seven months after operation, one patient had recurrence 34 months after surgery, 3 patients were uneventful with follow up duration from half a month to 32 months. Conclusion Adamantinoma occurring in long bones is very rare. The correct diagnosis requires adequate sample selection, careful morphologic observation, immunohistochemistry and molecular genetics.

Objective To explore cost effective means for early detection of malignant tumors in individuals undergoing health check-up.Methods This was a retrospective study involving 280,477 participants who had undergone health check-up including essential items from 2012 to 2016 at the Health Assessment and Intervention Research Center of Jiangsu Province.The protocol was composed of four steps.First,essential items were decided and conducted for all health examination participants.Second,cases with a high risk of malignant tumors were collected and additional tests were specified.Third,suspected malignant tumor cases were identified and recommendations for referrals and follow-up were made.Finally,physicians in charge of follow up would urge suspected cases to visit an oncologist,update case files,give regular instructions,and track recall results.Results There were 517 microscopically confirmed cases of malignant tumors,representing a detection rate of 184/100,000(1.84‰)in individuals seeking regular health check-up and of 2,023/100,000 (20.23‰)in those receiving follow ups.The five most prevalent malignant tumors were thyroid cancer (140 cases or 0.499‰),lung cancer(120 cases or 0.428‰),breast cancer(35 cases or 0.374‰),kidney cancer(55 cases or 0.196‰)and prostate cancer (33 cases or 0.177‰).Conclusions Essential examination items in combination with subsequent special tests,specialist referrals and follow ups are a cost effective way for early detection of malignant tumors in people seeking regular health examinations.

ObjectiveTo investigate the effects of Linggui Zhugantang on mitochondrial fission and fusion and silencing information regulator 3（Sirt3）/adenosine monophosphate dependent protein kinase （AMPK） signaling pathway in chronic heart failure （CHF） rats after myocardial infarction （MI）. MethodSD rats randomly divide into sham operation group （normal saline ，thread only without ligature）， model group （normal saline， ligation of the left anterior descending coronary artery proximal to the heart）， Linggui Zhugantang group （4.8 g·kg^-1） and Captopril group （0.002 57 g·kg^-1）， with 10 rats in each group. Administere drug continuously for 28 days. Echocardiography detected cardiac function parameters. Hematoxylin eosin （HE） staining observed the pathological changes of the heart. Immunofluorescence detected the levels of reactive oxygen species （ROS）. JC-1 detect mitochondrial membrane potential. Colorimetry measure adenosine triphosphate （ATP）， superoxide dismutase （SOD）， malondialdehyde （MDA）， mitochondrial respiratory chain complex activity （Ⅰ-Ⅳ）. TdT-mediated dUTP nick end labeling （TUNEL） staining detected the apoptosis rate of myocardial tissue. Western blot detected protein expression levels of Sirt3， phosphorylated AMPK （p-AMPK）， phosphorylated dynamic-related protein 1（p-Drp1）， mitochondrial fission protein 1（Fis1）， mitochondrial fission factor （MFF）， optic atrophy protein 1（OPA1）. ResultCompared to the sham group， the left ventricular end diastolic diameter （LVIDd） and left ventricular end systolic diameter （LVIDs） were significantly increased in model group （P<0.01）， while the left ventricular short axis shortening rate （LVFS） and left ventricular ejection fraction （LVEF） were significantly decreased （P<0.01）. There were inflammatory cell infiltration and obvious pathological injury in myocardial tissue. ROS， MDA levels and myocardial cell apoptosis rate were significantly increased （P<0.01）， SOD level， ATP content， and membrane potential were significantly decreased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） was significantly decreased （P<0.01）. Levels of p-Drp1， Fis1， MFF proteins were significantly up-regulated （P<0.01）， while Sirt3， p-AMPK， OPA1 proteins level were significantly down-regulated （P<0.01）. Compared with model group， LVIDd and LVIDs were significantly decreased （P<0.01）， LVEF and LVFS were significantly increased （P<0.01）. Inflammatory cell infiltration and pathological damage of myocardial tissue were significantly relieved. ROS， MDA levels and myocardial cell apoptosis rate were significantly decreased in Linggui Zhugantang group and Captopril group （P<0.01）， SOD level， ATP content， and membrane potential significantly increased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） increased significantly （P<0.01），and p-Drp1， Fis1， MFF protein levels were significantly down-regulated （P<0.01）， Sirt3， p-AMPK， OPA1 protein were significantly up-regulated （P<0.01）. ConclusionLinggui Zhugantang can alleviate oxidative stress and apoptosis damage of myocardial cells， maintain mitochondrial function stability， and its effect may be related to mitochondrial mitosis fusion and Sirt3/AMPK signaling pathway.