Objective To study the differentially expressed genes (DEG) during the differentiation of human induced pluripotent stem cells (hiPSC) and human embryonic stem cells (hESC) into pericytes and endothelial cells, and to identify key molecules and signaling pathways that may regulate this differentiation process. MethodshiPSC and hESC were selected and expanded using mTeSR medium. A "two-step method" was used to induce the differentiation of hiPSC and hESC into pericytes and endothelial cells. Pericytes were identified using immunofluorescence staining, while endothelial cells were isolated and identified using flow cytometry. Total RNA samples were extracted on days 0, 4, 7, and 10 of differentiation and consistently significant DEGs were screened. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis were performed on the screened DEGs. ResultsBoth hiPSCs and hESCs successfully differentiated into pericytes and endothelial cells under induction conditions. Transcriptome sequencing results showed that with the extension of differentiation time, the DEGs in hiPSCs and hESCs were significantly upregulated or downregulated, following a generally consistent trend. During the differentiation process, marker genes for pericytes and endothelial cells were significantly upregulated. A total of 491 persistent DEGs were detected in both hiPSC and hESC, with 164 unique to hiPSCs and 335 to hESCs, while 8 DEGs were co-expressed in both cell lines. Among these, SLC30A3, LCK, TNFRSF8, PRDM14, and GLB1L3 showed sustained downregulation, whereas CLEC18C, CLEC18B, and F2RL2 exhibited sustained upregulation. GO enrichment analysis revealed that DEGs with sustained upregulation were primarily enriched in terms related to neurogenesis, differentiation, and developmental proteins, while DEGs with sustained downregulation were enriched in terms related to membrane structure and phospholipid metabolic processes. KEGG pathway analysis showed that upregulated genes were primarily enriched in cancer-related pathways, pluripotency regulatory pathways, the Wnt signaling pathway, and the Hippo signaling pathway, whereas downregulated genes were predominantly enriched in metabolism-related pathways. ConclusionsDuring the differentiation of hiPSC and hESC into pericytes and endothelial cells, 8 DEGs exhibit sustained specific expression changes. These changes may promote pericyte and endothelial cell differentiation by activating the Wnt and Hippo pathways, inhibiting metabolic pathways, releasing the maintenance of stem cell pluripotency, affecting the cell cycle, and inhibiting cell proliferation.
【摘要】 目的 研究人類免疫缺陷病毒(HIV)感染者和獲得性免疫缺陷綜合癥(AIDS)患者CD4+T淋巴細胞數變化(ΔCD4+T)和外周血淋巴細胞總數變化(ΔTLC)的相關性。探討用ΔTLC預測ΔCD4+T在監測HIV/AIDS患者疾病進展以及高效抗逆轉錄病毒治療(HAART)療效的價值。 方法 回顧性分析2005〖CD3/5〗2008年確診的91例HIV/AIDS患者的臨床資料。 結果 ΔTLC與ΔCD4+T呈直線正相關(r=0809,Plt;001),好于TLC與CD4+T的相關性(r=0712,Plt;001)。分別用ΔTLC 170、330、630、910個/μL細胞預測ΔCD4+T 50、100、200、300個/μL細胞時具有較好的預測價值,各項評價指標符合率基本達到90%以上,顯著高于相同時間下用TLC預測CD4+T計數的價值。 結論 應用ΔTLC預測ΔCD4+T,可比TLC更加直觀、準確的反映HIV感染者疾病進展和評價AIDS患者HAART的療效。【Abstract 】Objective To assess the utility of total lymphocyte count (TLC) changes (ΔTLC) in place of TLC to predict the development of HIV/AIDS. To investigate the monitoring value of ΔCD4+T on progress of HIV/AIDS and HAART which predicted by ΔTLC. Methods Clinical data of 91 patiens with HIV/AIDS diagnosed from 2005 to 2009 were retrospectively analyzed. Results A linear correlation was found between the value of ΔTLC and the value of CD4+T changes(ΔCD4+T)(r=0809,Plt;001),which was better than the correlation between TLC and CD4+T (r=0712,Plt;001).Using ΔTLC as 170,330,630,910 cells/μL,respectively for forecasting ΔCD4+T as 50,100,200,300 cells/μL,respectively,had a better predictive value with the area under ROC curve near to 09,significantly higher than using TLC for predicting CD4+T counts. Conclusion ΔTLC is more accurate than TLC to reflect the development of HIV/AIDS.
OBJECTIVE This paper was to study the biological characteristics of the transformed human embryonic tendon cells, the relation between cell growth and culture conditions, and to compare these features with that of human embryonic tendon cells. METHODS The pts A58H plasmid had successfully used to transform a tendon cell line from human embryo in our past work. The human embryonic tendon cells and the transformed human embryonic tendon cells were cultured in vitro. In different culture conditions, the growth curve were drawn respectively. Population dependence and proliferation capability of the cells were investigated through plate cloning test and soft agar culture. The collagen secreted by cells was identified by immunohistochemical method. RESULTS In routine culture condition, the growth properties of the human embryonic tendon cell and transformed cells were almost identical. The growth properties of the transformed cells were not changed when the cells were frozen storage. There were changes of growth characteristics of the transformed cells when the culture temperature was changed. The transformed cells could subcultured continually and permanently. The proliferation capability of the transformed cells were ber than that of the human embryonic tendon cells. Moreover, the growth of the transformed cells was serum-dependent, and the phenomenon of contact inhibition was observed. The transformed cells were not able to grow on soft agar culture. They had the capacity of secreting collagen type I. CONCLUSION The transformed human embryonic tendon cells could be subcultured continually and permanently, and their growth could be controlled by changing their culture conditions and they had no malignant tendency in biological characteristics. They could be taken as an ideal experimental material for tendon engineering.
Objective To explore risk factors of stroke-associated pneumonia (SAP) for elderly stroke patients in ICU, and analyze the predictive value of human leukocyte antigen-DR (HLA-DR) on monocytes for SAP. Methods During January 2015 to August 2016, 155 elderly patients with stroke were recruited. The level of monocyte HLA-DR expression was measured after admission and the incidence of SAP was recorded. The risk factors for SAP were analyzed by univariate and multivariate analysis. ROC curve was drawn to analyze prognostic value of HLA-DR. Results SAP occurred in 75 cases with occurrence rate of 48.4%, including 42 early-onset cases and 33 later-onset cases. Age (OR=11.532), Glasgow Coma Scale (OR=7.124), dysphagia (OR=8.846), mechanical ventilation (OR=15.184), atrial fibrillation (OR=7.869), smoking history (OR=11.784), diabetes (OR=7.185) were independent risk factors (all P<0.05). The expression rate of monocyte HLA-DR in the SAP patients was significantly lower than those in the patients without SAP (allP<0.05). Through the ROC curve analysis, the expression rate of HLA-DR that below 78.65% was the optimum cut-off value for prediction of SAP with the area under ROC curve of 0.922, the sensitivity of 80.0% and the specificity of 85.0%. The sensitivity to predict early-onset SAP was 90.5% (38/42), and to predict later-onset SAP was 66.7% (22/33). Conclusions Age, severe coma, dysphagia, mechanical ventilation, atrial fibrillation, smoking history and diabetes are risk factors for SAP in elderly stroke patients in ICU. The detection of monocyte HLA-DR has reference value for early prediction of SAP especially for early-onset SAP with higher sensitivity.
【摘要】 目的 探討絕經期促性腺激素及氯米芬在促排卵治療中適宜的治療方法。 方法 2004年8月〖CD3/5〗2008年5月對80例不孕患者實施促排卵治療。測定血雌激素、黃體生成素水平、陰道B型超聲、子宮頸黏液評分及基礎體溫測定監測排卵,并觀察不良反應的發生情況。 結果 使用氯米芬及絕經期促性腺激素排卵率分別為773%和856%；卵巢過度刺激綜合癥發生率為150%,大多由使用絕經期促性腺激素方案引起,且起始劑量150 U；未破裂卵泡黃素化綜合征的發生率為90%。 結論 絕經期促性腺激素和氯米芬治療排卵障礙性不孕有較好的療效,絕經期促性腺激素和氯米芬促排卵治療效果與卵巢的狀態及激素水平有關。促性腺激素的使用應強調個體化,以達到較好的治療效果并降低卵巢過度刺激綜合癥的發生。【Abstract】 Objective To explore the proper method with human menopausal gonadotropin and clomiphene in facilitating ovulation treatment. Methods Eighty infertility patients with the facilitating ovulation treatment were included from August 2004 to May 2008. Ovulation monitoring was based on the level of estrogen and luteinizing hormone, transvaginal B ultrasound, the cervical Inlser score and assay of basal body temperature. Besides, adverse reactions were observed. Results The rates of ovulation of clomiphene and human menopausal gonadotropin were 77.3% and 85.6%. The rate of ovarian hyperstimulation syndrome (OHSS), which was mostly caused by human menopausal gonadotropin with 150 IU, was 150%. The rate of luteinized unruptured follicle syndrome (LUFS) was 90%. Conclusion Individual therapy with human menopausal gonadotropin and clomiphene is essential to infertility patients with ovulation barrier. The efficacy of human menopausal gonadotropin and clomiphene is relevant to the ovarian condition and the hormone levels. Individual using of hormone is important in the facilitating ovulation treatment, which is helpful to increase the effective efficacy and prevent the OHSS.
Objective To investigate the differentiation of theadipose-derived adult stem cell (ADASC) induced by the recombinant adenovirus’s containing fibers derived from B-group serotype 35 (rAd5/F35)mediated human bone morphogenetic protein 7 (hBMP-7) gene and to explore a new cell sourcefor the bone tissue engineering. Methods The hBMP-7 gene wasamplified with the pcDNA1.1/AMP-hBMP-7 plasmid as a formwork. After the purification, the gene fragment was cloned into the pDC316 carrier for the recombination of the plasmid of pDC316-hBMP-7. The 293 cells were cotransfected by the skeleton plasmid of pBHG-fiber5/35 and the shuttle plasmid of pDC316-hBMP-7, and the recombinant plasmid of Ad5/F35-hBMP-7 was obtained; the recombinant plasmid of Ad5/F35enhancd green fluorescent protein(EGFP) was obtained by the similar method. The rat ADASCs were cultured and transfected by the Ad5/F35-hBMP-7plasmid and the Ad5/F35-EGFP plasmid, respectively; the remaining untransfected ADASC were used as the controls. The morphology and the growth pattern of the transfected cells were evaluated. The transcription and the expression of the transfected genes and the steogenic phenotypes such as calcium nodules and osteocalcin were evaluated by ELISA. Results The identification of PCR and enzyme cutting showed that the construction of the recombinant Ad5/F35-hBMP-7 plasmid could be confirmed. The transfection rate of the ADASC by the Ad5/F35-EGFP plasmid was determined to be greater than 90%. The hBMP-7 gene in thetransfected ADASC could express the corresponding protein, and the formation ofthe calcium nodules could be found in the induced group. The electron microscopy showed that there was a calcium element in the cytoplasm, the alkaline phosphatase result was positive, and the expression of osteocalcin was increased. Conclusion The rAd5/F35-hBMP-7 gene can promote the differentiation of the adiposederived adult stem cells to the osteoblasts in the bone tissue engineering.
ObjectiveTo investigate the regulation of human bone marrow mesenchymal stem cells (hBMSCs) osteogenic and adipogenic differentiations mediated by Wnt10b adenoviral vector in vitro. MethodsThe hBMSCs from ilial bone tissue in adults at passage 4 were infected by Wnt10b gene expression adenoviral vector (group A), Wnt10b-shRNA adenoviral vector (group B), and empty vector (group C), and non-transfected hBMSCs served as the blank control group. Then the cells were cultured separately in the circumstance of osteogenic induction, adipogenic induction, and non-induction. The alkaline phosphatase (ALP) staining, alizarin red staining, and oil red O staining were used to detect the osteogenic and adipogenic differentiations; real-time fluorescent quantitative PCR and Western blot were used to analyze the expressions of osteoblast and adipocyte genes and proteins. ResultsThe results of ALP staining were positive after osteogenic induction, group A showed strong staining, and group B showed the weakest staining. The results of alizarin red staining showed that there were a lot of patchy confluent brown mineralized nodules in group A; a few punctate brown mineralized nodules were seen in group B; and many punctuate brown mineralized nodules were found in groups C and D. The results of oil red O staining showed strong staining in groups B, C, and D after adipogenic induction, especially in group B; scattered or small clustered staining was observed in group A. The expressions of osteoblast genes and proteins were significantly higher in group A than groups B, C, and D, and in groups C and D than group B by real-time fluorescent quantitative PCR and Western blot test; however, the expressions of adipocyte genes and proteins showed a contrary tendency. ConclusionThe high level expression of Wnt10b can enhance osteogenic differentiation of hBMSCs, and the low level expression of Wnt10b can increase adipogenic differentiation of hBMSCs.
ObjectiveTo study the characteristics of the human umbilical cord perivascular cells (HUCPVC) isolated from human first trimester umbilical cord perivascular layer tissues and the differentiation into islet-like cell clusters in vitro. MethodsThe HUCPVC derived from human first trimester umbilical cord which was donated by the volunteers were isolated and subcultured. The surface markers such as stage-specific embryonic antigen 1 (SSEA-1), SSEA-3, SSEA-4, OCT-4, TRA-1-60, and TRA-1-81 were detected by immunohistochemical method. The first trimester HUCPVC were induced to embryoid bodies (EB)-like cell aggregations and islet-like cell clusters in vitro through a simple stepwise culture protocol (5 steps). The expressions of specific markers[α-fetoprotein (AFP), Nestin, and smooth muscle actin (SMA)] were measured by immunohistochemical method; and the ability of glucose-stimulated insulin secretion was analyzed. ResultsThe first trimester HUCPVC were successfully isolated and could be passaged steadily more than 10 generations, which expressed SSEA-3, SSEA-4, OCT-4, TRA-1-61, and TRA-1-81. The first trimester HUCPVC were successfully induced into EB-like cell aggregations and islet-like cell clusters. The EB-like cell aggregations could express markers of three germ lineages:AFP, Nestin, and SMA. The islet-like cell clusters could release insulin significantly in response to elevated concentrations of glucose in vitro (t=7.444, P=0.002). The insulin contents were (23.2±5.3) mU/L and (7.0±0.5) mU/L in high and low glucose media, respectively. ConclusionThe first trimester HUCPVC has the ability to differentiate into islet-like cell clusters which can secret insulin in vitro.
Objective To analyze the factors associated with the adoption of targeted therapy in patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer and to generate evidence to inform decision-making on public security policy regarding innovative anticancer medicines for the benefit of patients. Methods The study population comprised female patients diagnosed with HER2-positive breast cancer and treated at Fujian Cancer Hospital from 2014 to 2020. The patients were eligible for targeted therapy. The demographic and sociological characteristics and clinical information of patients were extracted from the hospital information system. We performed binary logistic regression analysis of factors associated with the adoption of targeted therapy in patients with HER2-positive breast cancer. We also divided the participants into two groups according to their tumor stage for subgroup analysis. Results A total of 1 041 female patients with HER2-positive breast cancer were included, among them, 803 received targeted therapy. In September 2017, molecular-targeted medicines for HER2-positive breast cancer began to be included in the local basic health insurance program. Only 282 (35.1%) patients adopted targeted therapy before September 2017, after which this number increased to 521 (64.9%). Among the patients who adopted targeted therapy, most were formally employed (45.8%) and enrollees of the urban employee health insurance program (66.0%). Among those who did not adopt targeted therapy, most were unemployed (42.4%) and enrollees of the resident health insurance program (50.0%). Binary logistic regression analysis revealed that patient occupation, gene expression of estrogen receptor, tumor stage, surgery or not, radiotherapy or not, and undergoing treatment before or after September 2017 were correlated with the adoption of targeted therapy (P<0.05). Conclusions Inclusion of targeted medicines for HER2-positive breast cancer in the health insurance program substantially increased the overall administration of these therapies. Individual affordability is a critical factor associated with the application of targeted therapy in eligible patients. Future policies should enhance the public security of patients with a relatively weak ability to pay and provide insurance coverage for innovative anti-cancer medicines.
Objective To investigate the expression of human leukocyte antigen-G （HLA-G） in hepatocellular carcinoma （HCC） tissues， and to evaluate the prognosis of patients after liver transplantation. Methods The clinical data of 83 patients with HCC who underwent liver transplantation from January 2004 to May 2008 in the Liver Transplantation Center of the Third Affiliated Hospital of Sun Yat-sen University were analyzed retrospectively. The expression of HLA-G in HCC tissues was detected by using immunohistochemical analysis. The Kaplan-Meier method was used to evaluate the cumulative survival rate and tumor-free survival rate. Log-rank test and Cox regression model were used to analyze the single and muti-factor for tumor-free survival rate, respectively. Results Among the 83 patients，there were tumor recurrence in 35 patients （42.2%）. The 1-year，3-year, and 5-year of cumulative survival rate was 97.2%，89.8%, and 43.1%, respectively. The 1-year，3-year， and 5-year of tumor-free survival rate was 93.6%，68.9%, and 38.7%, respectively. The positive rate （68.7%） of HLA-G expression in HCC tissues was significantly higher than that in paracancerous tissues （15.7%）, P＜0.01. A significant association was found between expression of HLA-G and tumor size， vascular invasion, and pathology differentiation （P＜0.05）. Single factor analysis showed that the expression of HLA-G （P＜0.01）， tumor size （P＜0.05）， vascular invasion （P＜0.01）， and pathology differentiation （P＜0.01） effected on tumor-free survival rates of HCC patients after liver transplantation. The tumor-free survival rate in positive expression of HLA-G group was significantly lower than that in negative expression of HLA-G group （P＜0.01）. Cox regression model analysis showed that the expression of HLA-G （P＜0.05）， vascular invasion （P＜0.01）， and pathology differentiation （P＜0.05） were independent risk factors that affected the tumor-free survival rate of HCC patients after liver transplantation. Conclusions There is expression of HLA-G in HCC tissues. The independent risk factors that affecting the tumor-free survival rate of HCC patients after liver transplantation include the expression of HLA-G， vascular invasion, and pathological differentiation. Taking interferential measures for patients with positive expression of HLA-G and strict selection of indication of liver transplantation for HCC can reduce the recurrence rate of tumor.