AIM: To develop a simple, convenience, and inexpensive method on primary culture model of pancreatic islets in rats for the study of anti-diabetic drugs. METHODS: The pancreases of SD rats were separated from the pancreatic duct with cold Hank' s solution and picked. Then the pancreases were cut into pieces and repeatedly digested by collagenase at 37℃ for the short durations of the experiment. The isolated islets were identified by dithizone staining and the viability was evaluated by trypan blue staining. Pancreatic islets were incubated in RPMI 1640 or DMEM for 14 - 16 h, then they were transferred to new culture plates with the same medium mentioned above. Determination of insulin content of su-pernate and cell lysate and the experiment of insulin secretion by stimulation of glucose and implantation of micewith STZ-induced diabetes were used for evaluated the function of islets. RESULTS: The viability of isolated pancreatic islets was more than 95% and the purity of cultured islets was about 85% . The insulin synthesis, secretion and sensitivity of islets stimulate by glucose which were cultured in RPMI 1640 were higher than that in DMEM. The levels of blood glucose recovered to normal in type 1 diabetic mice after islets implantation. CONCLUSION : The islets got in this study have higher purity and viability with the normal biological activity for about 7 days by this method and they can be used as a cell model for the study of diabetes in vitro .

OBJECTIVE: To study the mechanism of Huoxue Qianyang Granule (HXQYG), a traditional Chinese compound medicine, in revising the left ventricular hypertrophy of hypertension. METHODS: Spontaneous hypertension rats (SHR) were randomly divided into seven groups: untreated group, Songling Xuemaikang (SLXMK)-treated group, captopril-treated group, high-, medium- and low-dose HXQYG-treated groups, and normal control group. The systolic blood pressure (SBP) and left ventricular mass index (LVMI) were measured. The content of angiotensin II (Ang II) in left ventricular tissue was determined by radioimmunoassay. The expressions of angiotensin converting enzyme (ACE) protein and mRNA in left ventricular tissue were analyzed separately by immunohistochemical method and RT-PCR. RESULTS: (1) SBP and LVMI were higher in the untreated group than those in the normal control group, and they were lower in the high- and medium-dose HXQYG-treated groups than those in the untreated group, but higher than those in the captopril-treated group, and without significant difference as compared to those in the SLXMK-treated group. (2) The content of Ang II and expressions of ACE protein and mRNA in the left ventricular tissue in the untreated group were higher than those in the normal control group, and they were lower in the HXQYG-treated groups than those in the untreated group, but higher than those in the captopril-treated group, and without significant difference as compared to those in the SLXMK-treated group. CONCLUSION: HXQYG can reverse the left ventricular hypertrophy of SHR, which may be due to decreasing the amount of Ang II and expressions of ACE protein and mRNA in the left ventricular tissue.

Objective To investigate the expression of HMGB1 mRNA in endometrial carcinoma and its correlation with clinicopathological features of endometrial carcinoma. Methods Semi-quantitative RT-PCR method was used to detect the expression level of HMGB1 gene in 56 cases of endometrial carcinoma and 20 cases of normal endometrium. Results The expression of HMGB1 mRNA in endometrial carcinoma (0. 3512 ± 0. 0985 ) was significantly higher than that in normal endometrium (0. 2208 ± 0. 0170 ).There was a significant difference in the two groups( P ＜0. 05 ). Grouping by clinicopathological features,the expression of HMGB 1 mRNA in endometrial carcinoma had a correlation with clinical-surgical stage ( P＜0. 05), metastasis of lymph nodes ( P ＜ 0. 05), and depth of myometrial invasion ( P ＜ 0. 05 ). Conclusion The high level of HMGB1 mRNA indicated that HMGB1 might play an essential role in the genesis, growth, invasion and metastasis of endometrial carcinoma.

Objective To explore the effect of endothelial progenitor cells (EPCs) on the proliferation of co-cultured rat vascular smooth muscle cells(VSMCs). Methods Mononuclear cells were isolated from fresh cord blood by 6% hydroxyethyl starch (HES) and density gradient centrifugation. Isolated mononuclear cells were cultured in EGM-2 medium supplemented with 20% fetal bovine serum (FBS), VEGF,bFGF and other growth factors. Biological features of EPCs were observed at different time points, and EPCs were identified by morphology, fluorescence double-staining and flow cytometry. Indirect immunofluorescence was performed to analyze the expression of α-SM-actin, calponin of VSMCs special antigen. Co-culture system of EPCs and VSMCs was established by transwell permeable support. FBS (20%) was used to stimulate the proliferation of VSMCs. In a VSMCs/EPCs co-culture system, the DNA synthesis ability, total protein level and cell cycle of VSMCs were determined by BrdU marking method,protein quantitation and flow cytometry after co-culture for 6, 12, 24,48 and 72 h. Results After co-culture for 12, 24, 48, and 72 h, the DNA synthesis ability and total protein level of VSMCs significantly decreased compared with the control group(P＜0.05). Flow cytometry showed that the percentage of S phase of VSMCs in VSMCs/EPCs co-cultured group significantly decreased and the percentage of G_1 phase increased markedly compared with the control group(P＜0.05). The maximal inhibitory effect was observed at 48 h. Conclusion Early EPCs could inhibit the proliferation of VSMCs.

The aim of this study was to investigate the effect of Paris saponin I (PS I) on human gastric carcinoma cell growth and apoptosis and to explore the potential mechanisms. The proliferation of SGC7901 cells was monitored by the MTT cell viability assay, while the nuclear morphology of apoptotic cells was assessed by Hoechst 33258 staining. Flow cytometry was performed to analyze the cell cycle progression of propidium iodide (PI)-stained SGC7901 cells and the apoptotic rate of annexin V/PI-stained cells. Western blotting was used to examine the expression of several cell cycle proteins, including cyclin B1 and Cdk1, and the apoptosis-regulated proteins Bcl-2, Bax, cytochrome c, procaspase-9, and procaspase-3. The MTT assay demonstrated that PS I could induce significant dose- and time-dependent inhibition of SGC7901 cell proliferation. Marked morphological changes, including condensation of chromatin, nuclear fragmentation and apoptotic bodies were clearly shown on Hoechst 33258 staining. PSI treatment also resulted in the disruption of the cell cycle at G(2)/M and the induction of apoptosis. Following PSI treatment, the cell cycle-related proteins cyclin B1 and Cdk1 were down-regulated. Expression of the pro-apoptotic protein Bax was increased, while anti-apoptotic protein Bcl-2 decreased. PSI treatment resulted in elevated cytoplasmic cytochrome c and activation of the apoptotic proteases caspase-9 and caspase-3. These data indicate that PS acts as an inhibitor of proli I feration in SGC7901 cells by inducing cell cycle arrest and mitochondria-dependent apoptosis. PSI is a potential therapeutic agent against human gastric carcinoma.

Objective To investigate the relationship between hyperlipidemia and hyperuricemia.Methods From February 1,2012 to May 31,2017,a physical examination queue for serving and retired employees in Xiangya Hospital was established.As the survey's baseline,height,weight,waist circumference,blood lipids,blood pressure,blood glucose,creatinine,and serum uric acid were collected.The normal group was the control group,and the dyslipidemia group was the exposure group.The occurrence of hyperuricemia was investigated during follow-up.A multivariate Cox proportional hazards regression model was used to analyze hyperlipidemia.Four different clinical types (hypercholesterolemia,hypertriglyceridemia,mixed hyperlipidemia,and low high-density lipoprotein hyperlipidemia) and hyperuricemia had an incidence of hazard ratio (HR) and confidence interval (95％CI).Results A total of 1 553 people entered the follow-up cohort.A total of 5 297 patients were followed up for an average of 3.4 years.Three hundred and ninety-four cases of hyperuricemia were collected.The density of hyperuricemia was 744/10 000 years.The hyperuricemia group was followed up for 2 509 years,with hyperuricemia occurring in 142 cases,and hyperuricemia in the hyperlipidemia group of 566/million years.The hypercholesterolemia,high triglyceride,mixed hyperlipidemia,and low-density lipoprotein groups were followed up for 1 431,403,580,92 years,respectively,and high uric acid occurred respectively.In 105,64,72 and 11 cases,the incidence of disease was 734/million years,1 588/million years,1 241/million years,1 196/million years;the difference was statistically significant (P＜ 0.01).Hyperlipidemia and its four clinical types,hypercholesterolemia,hypertriglyceridemia,mixed hyperlipidemia,and hypodic lipoproteinemia,were associated with hyperuricemia.HR (95％CI) was 1.971 (1.604-2.421),1.441 (1.120-1.855),3.103 (2.309-4.169),2.434 (1.833-3.233),2.336,respectively.(1.265-4.316),P＜ 0.01;after adjusting the influence of age,sex,body mass index,hypertension,hyperglycemia,and hyper creatinine,HR (95％CI) was 1.885 (1.533-2.317),1.450 (1.127-1.866),2.881 (2.141-3.876),2.118 (1.588-2.825),2.451 (1.326-4.528) P＜0.01.Conclusions Hyperlipidemia and its four different clinical types (hypercholesterolemia,hypertriglyceridemia,mixed hyperlipidemia,and low-density lipoproteinemia) are all associated with the onset of hyperuricemia.

The aim of this study was to investigate the effect of Paris saponin Ⅰ (PS Ⅰ ) on human gastric carcinoma cell growth and apoptosis and to explore the potential mechanisms.The proliferation of SGC7901 cells was monitored by the MTT cell viability assay,while the nuclear morphology of apoptotic cells was assessed by Hoechst 33258 staining.Flow cytometry was performed to analyze the cell cycle progression of propidium iodide (PI)-stained SGC7901 cells and the apoptotic rate of annexin V/PI-stained cells.Western blotting was used to examine the expression of several cell cycle proteins,including cyclin B1 and Cdkl,and the apoptosis-regulated proteins Bcl-2,Bax,cytochrome c,procaspase-9,and procaspase-3.The MTT assay demonstrated that PSⅠ could induce significant doseand time-dependent inhibition of SGC7901 cell proliferation.Marked morphological changes,including condensation of chromatin,nuclear fragmentation and apoptotic bodies were clearly shown on Hoechst 33258 staining.PSⅠ treatment also resulted in the disruption of the cell cycle at G2/M and the induction of apoptosis.Following PSⅠ treatment,the cell cycle-related proteins cyclin B 1 and Cdk1 were downregulated.Expression of the pro-apoptotic protein Bax was increased,while anti-apoptotic protein Bcl-2decreased.PSⅠ treatment resulted in elevated cytoplasmic cytochrome c and activation of the apoptotic proteases caspase-9 and caspase-3.These data indicate that PSⅠ acts as an inhibitor of proliferation in SGC7901 cells by inducing cell cycle arrest and mitochondria-dependent apoptosis.PSⅠ is a potential therapeutic agent against human gastric carcinoma.

OBJECTIVE: To purify and identify HMGB1 secreted by liver cells HepG2 and immune cells U937. METHODS: We cultured the liver cell lines HepG2 and immune cell lines U937, and stimulated them with HMGB1 (400 ng/mL) for 20 h. Then the supernatant was collected. Ultrafiltration centrifugation, CM-Sepharose cation, DEAE-Sepharose anion exchange chromatography, Sephadex G75-gel filtration chromatography, and immunoprecipitation were used for purification. The molecular weight and identity of HMGB1 was confirmed by SDS-PAGE and Western blot. RESULTS: A sharp stained protein band with a molecular weight of about 26 kD was obtained by SDS-PAGE analysis and shown to be HMGB1 confirmed by Western blot. CONCLUSION High purified HMGB1 can be separated from these two cell lines.
Cell Culture Techniques ; Electrophoresis, Polyacrylamide Gel ; methods ; HMGB1 Protein ; isolation & purification ; metabolism ; Hep G2 Cells ; Hepatocytes ; metabolism ; Humans ; Monocytes ; metabolism ; U937 Cells

Abstract: Objective　To evaluate the free thalassaemia screening programme for preconception and pregnancy in Hainan Province, and to provide a theoretical basis for optimizing the screening process for thalassaemia. Methods From November 2020 to July 2021, a survey was conducted on 10 396 adults with Hainan household registration who participated in the Epidemiological Survey of Thalassemia in Hainan Residents in 19 cities and counties of Hainan Province. All of them underwent routine blood tests, haemoglobin electrophoresis tests and genetic tests for thalassaemia. The optimal diagnostic cut-off values for mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and haemoglobin adult type 2 (HbA2) were determined using screening test indexes such as receiver operating characteristic curve and sensitivity. The diagnostic effectiveness of different primary screening programs for thalassemia gene carriers was evaluated. Results Using the existing MCV single-indicator thalassemia primary screening protocol in Hainan Province, where individuals with MCV<82 fL undergo thalassemia gene testing, resulted in a high missed diagnosis rate (34.06%) and low sensitivity (65.94%). The optimal cut-off values for MCV screening for alpha-and beta-thalassaemia were 84.45 fL and 79.05 fL, respectively; the optimal cut-off values for MCH screening for alpha-and beta-thalassaemia were 27.95 pg and 25.15 pg, respectively. The optimal cut-off value for HbA2 screening for alpha-thalassaemia was less than 2.55% and greater than 3.35% for beta-thalassaemia. The "combined HbA2 or MCH or MCV screening protocol" with the cut-off values recommended in this study had a better performance in primary screening for thalassemia, with the highest sensitivity (92.96%) and negative predictive value (92.67%) and the lowest underdiagnosis rate (7.04%), statistically significant differences compared with the existing protocol (P<0.05). Conclusions The current process of screening for thalassemia in Hainan Province may lead to missed diagnoses. The combined use of MCV, MCH and HbA2 for thalassemia screening, adopting locally suitable cutoff values for primary screening indicators, can improve the incidence of missed reporting of thalassemia and enhance diagnostic effectiveness.

Objective:To investigate the correlation between hyperhomocysteinemia (HCA) and hyperuricemia (HUA).Methods:A total of 8 465 individuals who received a medical examination at the Xiangya Hospital Health Management Center of Central South University were selected as research subjects from January to June 2018. The multi-factor binary Logic regression method was adopted and four different models. The ratio ( OR) and its 95% trusted interval ( CI) between Hhcy group and normal control group were analyzed. Results:Compared to the normal control group, the OR(95% CI) of the probability of HUA in the Hhcy population was 3.272 (95% CI: 2.839 to 3.771, P<0.001) without correcting other factors; After correcting the influence of age and gender, the OR (95% CI) of the Hhcy population with HUA probability was 2.111 (95% CI: 1.811-2.461, P<0.001); After correcting age, gender, Body Mass Index (BMI), blood pressure, fasting blood sugar, triglycerin, low-density lipoprotein, and high-density lipoprotein, the OR(95% CI) of the Hhcy population with HUA probability was 2.163 (95% CI: 1.845 to 2.535, P<0.001); After correcting age, sex, BMI, blood pressure, fasting blood sugar, triglycerin, low-density lipoprotein, high-density lipoprotein, drinking, smoking, exercise time, and exercise intensity, the OR (95% CI) of the Hhcy population had a HUA probability of 2.209(95% CI: 1.845 to 2.646, P<0.001). Conclusion:The cross-sectional study confirmed that Hhcy was positively correlated with HUA. Reducing homocysteine levels may be a new approach to the health management of HUA.