In this study, we aimed to investigate the influences of conditioned medium from human umbilical vein endothelial cells (HUVEC) on cancer stem cell phenotype of human hepatoma cells. HUVEC and human hepatoma cells (MHCC97H) were cultured, respectively, and then the MHCC97H cells were co-cultured with conditioned medium from HUVEC (EC-CM) with Transwell system. Anti-cancer drug sensitivity, colony-formation, migration/invasion ability, expression of cancer stem cell marker and sphere formation were performed to determine the cancer stem cell phenotype in MHCC97H cells. We found that MHCC97H cells co-cultured with EC-CM exhibited significantly higher colony-formation ability and lower sensitivity of anti-cancer drugs 5-FU and Cis. Transwell assay showed that treatment with EC-CM obviously increased migration and invasion of MHCC97H cells. Moreover, increased sphere forming capability and expression of CD133 in MHCC97H cells were observed after co-cultured with EC-CM. These results suggested that EC-CM could promote cancer stem cell phenotype of hepatoma cells.
Antineoplastic Agents ; pharmacology ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Coculture Techniques ; Culture Media, Conditioned ; Fluorouracil ; pharmacology ; Human Umbilical Vein Endothelial Cells ; chemistry ; Humans ; Liver Neoplasms ; Neoplastic Stem Cells ; cytology ; Phenotype

With the incidence of type 2 diabetes mellitus increasing year after year, the technology of drug screening of type 2 diabetes mellitus progress rapidly, from the level of animal screening to cellular and molecular screening model, from the traditional drug screening technology to high efficient and throughput screening. This paper will summarize the technology of drug screening in the therapy of type 2 diabetes mellitus.
Animals ; Diabetes Mellitus, Type 2 ; drug therapy ; genetics ; Drug Evaluation, Preclinical ; methods ; Humans ; In Vitro Techniques

BACKGROUND:Cel cryopreservation is required for clinical use of stem cel s, and the current process of cryopreservation however may be harmful to cel viability, pluripotency and differentiation capacity.
OBJECTIVE:To explore the effect of fructose and dithiothreitol on pluripotency and osteogenesis of cryopreserved bone marrow mesenchymal stem cel s.
METHODS:Bone marrow mesenchymal stem cel s were isolated from the bone marrow of Sprague-Dawley rats and pretreated with fructose (200μmol/L), dithiothreitol (500μmol/L) or combined components before cryopreservation. Then the cel s were cryopreseved for 6 months and the morphology of cel s was observed by inverted microscopy. The cel viability was evaluated by MTT, and real-time PCR was used to detect the mRNA expression of Nanog, OCT4 and Sox2. Alkaline phophatase activity assay and alizarin red staining were utilized to detect the osteogenic capacity of bone marrow mesenchymal stem cel s.
RESULTS AND CONCLUSION:Images captured by inverted microscopy showed no significant difference in cel morphology between groups. The MTT results indicated that fructose and combined pretreatment could promote the cel viability of bone marrow mesenchymal stem cel s after cryopreservation, while the real-time PCR results demonstrated that dithiothreitol significantly facilitated the expression of Naogo and Sox2 in bone marrow mesenchymal stem cel s. Moreover, ALP activity assay and alizarin red staining confirmed the positive effects of fructose, dithiothreitol and combined pretreatment on osteogenic capacity of bone marrow mesenchymal stem cel s after cryopreservation, and the best effects were found after pretreatment with dithiothreitol and combined components. Overal , these findings indicate that fructose pretreatment is beneficial for cel viability of cryopreseved bone marrow mesenchymal stem cel s, and dithiothreitol contributes to maintaining the pluripotency and osteogenesis capacity of cryopreseved bone marrow mesenchymal stem cel s.

Bone marrow derived mesenchymal stem cell (BMSC) is one of the crucial cell types which plays roles in wound healing of tissues. In the last decades, it was believed that BMSCs promoted wound healing by differentiating into multiple lineages and placing the wounded tissues. In recent years, a new viewpoint arose from evidences that the paracrine effect of BMSCs might play a more important role in the process of wound healing than differentiation. Understanding the role of BMSCs paracrine in wound healing would be vital to clarify the mechanism how BMSCs take part into the process of wound healing. In this paper, we review the new research processes of BMSCs paracrine in wound healing of tissues.
Animals ; Bone Marrow Cells ; cytology ; physiology ; Cell Differentiation ; physiology ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; physiology ; Paracrine Communication ; physiology ; Wound Healing ; physiology

In our study, a two-phase culture system was developed to acquire large amount of CD41+ and polyploidy cells. Human mobilized peripheral blood CD34+ (PB CD34+) cells were first cultured in expansion medium (Cocktail or CC100 medium) for 3,4,5 or 6 days, and then cultured in megakaryocytic differentiation medium containing TPO and SCF for additional 7, 8 or 9 days. Cell expansion, morphology, CD41+ cell percentage and DNA content were investigated to evaluate the protocol. The result showed that more CD41+ and polyploidy cells could be obtained following the two-phase culture with Cocktail medium than with CC100. Moreover, with 3 days expansion in Cocktail medium plus 7 days in differentiation medium, the initial CD 34+ cells obtained 16-fold expansion of CD41+ cells and 3-fold expansion of polyploidy cells, such obtained level being significantly higher than that of culturing cells with only one step in TPO or TPO+SCF. We conclude that with the two-phase culture system, PB CD34+ cells can expand and differentiate to more CD41+ and polyploidy cells than those cultured only in accordance to the one-stage culture protocol, so a new and highly efficient megakaryocyte differentiation model for megakaryocyte and platelet related researches is provided already.
Antigens, CD34 ; blood ; Blood Cells ; cytology ; Cell Culture Techniques ; methods ; Cell Differentiation ; physiology ; Colony-Stimulating Factors ; physiology ; Hematopoietic Stem Cell Mobilization ; methods ; Humans ; Megakaryocytes ; cytology ; Stem Cells ; cytology

Hepatocellular carcinoma (HCC) is one of the most common human malignant tumors worldwide; it is also hard to prevent its metastasis and recurrence by traditional treatments. Up to now, how to prevent and treat HCC is still a challenging problem in clinic. Cancer stem cells (CSCs) are cells within malignant tumor that possess the capacity to self-renew and differentiate to lead to the heterogeneous lineages of cancer cells that comprise the tumor, and are the root to cause metastasis, recurrence and bad prognosis of the cancer. Targeting CSCs is a novel therapeutic strategy for management and treatment of the cancer. In recent years, targeting intervention on liver cancer stem cells (LCSCs) gradually became a novel strategy for HCC treatment, and some exciting research results in the treatment of HCC were also achieved. In this review, we introduce the biological characteristics of LCSCs and highlight the therapeutic strategies for hepatocellular carcinoma by targeting intervention on LCSCs.
Animals ; Antineoplastic Agents ; therapeutic use ; Carcinoma, Hepatocellular ; therapy ; Humans ; Liver Neoplasms ; therapy ; Molecular Targeted Therapy ; Neoplastic Stem Cells ; metabolism ; pathology

Objective To investigate the influences of different matrix stiffness on proliferation ability and glucose metabolism of hepatocellular carcinoma (HCC) cells and to explore the correlation between metabolism and biological behavior changes of HCC cells resulted from the stiffness of extracellular matrix (ECM)．Methods The proliferation changes of HepG2 cells cultured on matrix with different stiffness were detected by CCK-8 assay and cell count assay. 2-NBDG and flow cytometry were used to detect the effect of matrix stiffness on glucose uptake. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of Glut1. Then, 2-DG was used to inhibit glycolysis, and the influences of matrix stiffness on proliferation of HepG2 cells were detected. Results The proliferation ability, glucose uptake and the expression of Glut1 of HepG2 cells increased with the matrix stiffness increasing. When glycolysis was inhibited, the proliferation ability of HepG2 cells grown on matrix with different stiffness was similar. Conclusions The mechanical microenvironment had an important effect on proliferation of HCC cells; matrix with a larger stiffness might promote proliferation of HCC cells through regulating glycolysis. The research findings provide a corresponding experimental basis for the clinical treatment of HCC cells and drug development targeting glucose metabolism.

Objective To investigate the regulatory effect of Xuebijing injection on inflammatory reaction during the preservation of isolated empty beating pig hearts with extracorporeal membrane oxygenation. Methods Twelve healthy Guangxi Bama miniature pigs were randomly divided into the Xuebijing group (n=6) and normal saline group (n=6). After the models were established in the Xuebijing group, Xuebijing injection was given at a dose of 5 mL/h through micropump in membrane oxygenator. In the normal saline group, an equivalent amount of 0.9% sodium chloride injection was pumped. Continuous pumping was performed for 8 h in both groups. The time of cardiac resuscitation and perfusion pressure, heart rate, perfusion flow rate after 8 h preservation were recorded in two groups. Pathological and ultrastructural changes of myocardial tissues in the left ventricular wall of hearts with cardiac arrest were observed after 8 h preservation. Serum levels of myocardial injury markers and inflammatory cytokines were detected in two groups at the beginning (T0), 2 h (T2), 4 h (T4), 6 h (T6) and 8 h (T8) after model establishment, respectively. The expression levels of NOD-like receptor protein 3(NLRP3), cysteinyl aspartate specific proteinase-1(Caspase-1), apoptosis-associated speck-like protein containing a CARD(ASC) messenger RNA (mRNA) in myocardial tissues were measured at T0, T2, T4, T6 and T8, respectively. Results There were no significant differences in the time of cardiac resuscitation and perfusion pressure, heart rate, perfusion flow rate after 8 h preservation between two groups (all P>0.05). Compared with the normal saline group, the levels of lactate dehydrogenase (LDH) at T4, creatine kinase (CK), LDH and α-hydroxybutyrate dehydrogenase (α-HBDH) at T6 and T8, tumor necrosis factor (TNF)-α at T4, T6 and T8, and interleukin (IL)-6, IL-18 and IL-1β at T0, T2, T4, T6 and T8 were lower, and the mRNA relative expression levels of NLRP3 and Caspase-1 at T2, T4 and T6, and Caspase-1 and ASC at T8 were lower in the Xuebijing group, respectively (all P<0.05). Hematoxylin-eosin staining and transmission electron microscopy showed that the degree of myocardial injury in the Xuebijing group was slighter than that in the normal saline group. Conclusions Xuebijing injection may effectively mitigate inflammatory response and exert certain myocardial protection effect during the ECMO preservation of isolated empty beating pig hearts.

Microgravity is a typical feature of the space. A large number of space flights and foundation simulation experiments have shown that cells show typical biological characteristics of aging, such as reduced cell proliferation and cell cycle arrest under microgravity or simulated microgravity. However, the molecular mechanism by which microgravity or simulated microgravity affects cellular senescence is not well understood. Understanding the mechanism controlling cellular senescence induced by microgravity environment is helpful for exploring anti-aging strategies and targeted interventions in space. In recent years, domestic and foreign scholars have carried out a number of researches and explorations on the effect of microgravity and simulated microgravity on cellular senescence as well as the related mechanisms. In this review, the latest research progress of this filed was summarized.

Objective:To analyze the influencing factors of abnormal 15-minute retention rate of indocyanine green (ICG R15) (≥10%) in patients with hepatocellular carcinoma, and to construct a nomogram model, and to evaluate the prediction efficiency of the nomogram model.Methods:The clinical data of 190 patients with hepatocellular carcinoma in Zhengzhou University People's Hospital from December 2017 to June 2022 were retrospectively analyzed, including 148 males and 42 females, aged (57.8±9.9) years. According to ICG R15, the patients were divided into ICG R15 normal group ( n=134, ICG R15<10%) and ICG R15 abnormal group ( n=56, ICG R15≥10%). Univariate and multivariate logistic regression were used to analyze the influencing factors of abnormal ICG R15, and the nomogram model was established. The predictive ability of the model was evaluated by receiver operating characteristic (ROC) curve and C-index, and the model was verified by calibration curve and decision analysis curve. Results:Abnormal ICG R15 group the proportion of liver cirrhosis, albumin ≤35 g/L, hemoglobin ≤110 g/L, platelet count ≤100×10 9/L, prothrombin time >13 s, alanine aminotransferase >40 U/L, aspartate aminotransferase >40 U/L, total bilirubin >34.2 μmol/L, and the largest tumor diameter >5.0 cm, spleen volume >383.1 cm 3, spleen volume to of non-tumor liver volume (SNLR) >0.276 and liver tumor volume >117.2 cm 3 were higher than that of ICG R15 normal group, and the differences were statistically significant (all P<0.05). Logistic regression analysis showed that liver cirrhosis ( OR=3.89, 95% CI: 1.28-11.80, P=0.016), spleen volume >383.1 cm 3( OR=5.17, 95% CI: 1.38-19.38, P=0.015), SNLR >0.276 ( OR=5.54, 95% CI: 1.44-21.26, P=0.013) and total bilirubin >34.2 μmol/L( OR=10.20, 95% CI: 1.88-55.39, P=0.007) increased the risk of abnormal ICG R15. A nomogram model was constructed based on the above risk factors. The C-index of the model was 0.915 (95% CI: 0.872-0.957), and the area under the ROC curve predicted by the nomogram model was 0.915 (95% CI: 0.871-0.958). The calibration curve showed that the correlation index of the abnormal ICG R15 predicted by the nomogram was similar to actual situation. Decision analysis curve showed high returns. Conclusion:Liver cirrhosis, spleen volume >383.1 cm 3, SNLR>0.276 and total bilirubin >34.2 μmol/L were indepentlent risk factors for abnormal ICG R15 in patients with hepatocellur carcinoma. The clinical prediction model of ICG R15 abnormality constructed by nomogram has good prediction efficiency, which can provide a reference for evaluating preoperative liver reserve function of patients with hepatocellular carcinoma.