Objective To investigate the method to isolate and culture hepatic stellate cells ( HSCs) for studying the cellular mechanisms of hepatic frbrosis.Methods HSCs were isolated by nycodenz density gradient centrifugation after the hepatocytes obtained from adult male gebils were digested with pronase, collagenase and DNase, infused via portal vein.The cell viability was determined by trypan blue exclusion test.The purity of HSCs was identified by detectingα-SMA, desmin immunohistochemical staining.Results The yield rate of HSCs was 0.5~1 ×107 per gerbil liver, and the cell viability was more than 90%.The percentage ofα-SMA-positive cells was more than 75%after 3 days primary culture and almost 100% cells were α-SMA and desmin positive in passage culture.Conclusion The successful protocol of primary culture of Mongolian gerbil HSC provide a technical support for research of relevant liver diseases and drug development in the future.

Objective To establish a fluorescence quantitative Taqman-PCR method for rapid and accurate detection of mouse poxvirus.Methods After sequence alignment and comparison, ERPV_027 gene was selected as the primer and probe design gene.Furthermore, the specificity, sensitivity, stability and reproducibility of these primers and probes were detected.Results The detection limitation of this method was 68 copies/μL.Data showed that this method has high specificity, which specifically amplifies mouse poxvirus, with no amplification signal of mouse hepatitis virus, Sendai virus, Salmonella and some other viruses and bacteria.This method also showed good stability and reproducibility. Conclusions This study has successfully established a fluorescence quantitative Taqman-PCR method for detection of mouse poxvirus, with high specificity, sensitivity, good stability and reproducibility, and a broad application potential.

Objective: To discuss the correlation between myeloid derived suppressor cells (MDSCs) and hepatic trans-planted tumor and to explore new ways to inhibit the development of hepatic cancer. Methods: We established the animal models with H_(22) hepatic carcinoma cells transplanted to the anterior right limb. Then the MDSCs morphology was observed with confocal microscopy and the proportion of MDSCs in blood and spleen was measured with flow cytometry. The 36 mice were divided into three groups: the control group, the low-dose group (2mg/kg) and the high-dose group (4mg/kg). Then As_2O_3 was injected twice a week to the mice before repeating the aforementioned measures. The direct effects of As_2O_3 on MDSCs cultured with H_(22)-ascites supernatant was observed. Results: At 25 days after transplantion, the tumor weight was increased to 5.67g, and the proportion of MDSCs in blood and spleen was increased to 20.46% and 9.50%, re-spectively. There was a positive correlation between hepatic transplanted tumor and MDSCs in blood and spleen and the relative factors were 0.95 and 0.96, respectively (t=-5.270 and 5.939, P<0.05). With the effect of As_2O_3, the proportion of MD-SCs in blood in low-dose group and high-dose group was 11.31% and 10.00% at 28 days after treatment, lower than that in the control group (t=3.193 and 5.486, P<0.05), and there was also a statistical difference between the high-dose group and low-dose group (t=3.066, P<0.05). The proportion of MDSCs in the spleen in low-dose group and high-dose group was 10.90% and 9.04% at 28 days, lower than that in the control group (t=3.586.and 5.279, P<0.05), but there was no statistical difference between the high-dose group and low-dose group (1=1.298, P>0.05). In vitro, the proportion of MDSCs in nutrient fluid was increased to 12.67% at 12 days after treatment with H_(22)-ascites supematant, and was decreased to 7.44% at 18 days after treatment with As_2O_3. Conclusion: The proportion of MDSCs in H_(22) tumor-bearing mice is increased because of tu-mor development. There is a positive correlation between MDSCs and hepatic transplanted tumor. As_2O_3 can decrease MD-SCs and inhibit tumor growth.

OBJECTIVETo explore the prophylaxis effect of pretreatment of allograft with methoxypolyethylene glycol-succinimidyl-propionic acid ester (mPEG-SPA) and anti-OX40L monoclonal antibody (McAb) on acute graft-versus-host disease (aGVHD) after allogeneic bone marrow transplantation (allo-BMT) in mice.

METHODSResponder splenocytes from C57BL/6 donor mice (H-2(b)) were co-cultured with stimulator splenocytes from BALB/c recipient mice (H-2(d)) for 7 days in the presence or absence of anti-OX40L McAb followed by mPEG-SPA chemical modification. Donor bone marrow cells plus the mixed culture of T-cells were then transplanted into lethally irradiated BALB/c mice. The BALB/c recipient mice were divided into four groups: group A (allo-BMT control group), group B(mPEG-SPA modification group), group C (anti-OX40L McAb pretreated group) and group D (mPEG-SPA and anti-OX40L McAb dual-treated group). Survival time and survival rate of the recipients were observed after allo-BMT. GVHD was assessed by clinical signs and histological changes of skin, liver and small intestines. Enzyme-linked immunosorbent assay (ELISA) was used to detect cytokines (IL-4, IL-10 and INF-gamma) production. Flow cytometry (FCM) analysis was used to detect allogeneic chimerism.

RESULTS(1) The mice in group A developed typical clinical signs of aGVHD and all mice died within 17 days after BMT with an average survival time (AST) of (12.1 +/- 5.5) days. The signs of aGVHD were less evident in mice of groups B, C and D, and their AST (36.2 +/- 24.9, 32.0 +/- 24.8 and 44.3 +/- 23.2 days, respectively) were all longer than that in group A (P < 0.05). AST of group D being the longest (P < 0.05). The survival rates at day 60 post-BMT in groups B, C and D were 50%, 41.7% and 66.7%, respectively. (2) Serum IFN-gamma level was increased after BMT in group A, and peaked in day 10 to day 15 post-BMT, while the level was decreased in groups B, C and D, reached the nadir on the day 10 post-BMT, with the lowest in group D (P < 0.01). After BMT, IL-4 and IL-10 levels were slightly decreased in group A, their levels were elevated in groups B and C (P < 0.05) and even more significantly increased in group D (P < 0.01). IL-4 and IL-10 levels peaked between day 10 and 15 post-BMT. (3) The average proportion of H-2(b) positive cells in recipient mice was 95% - 100% on day 60 post-BMT, with complete donor-type implantation.

CONCLUSIONCombination of mPEG-SPA and anti-OX40L McAb can block T-cell activated antigens and co-stimulatory pathway, regulate the T cells differentiation and induce the immune shift of Th0 cells toward Th2 cells. The immune tolerance induced by this method can significantly relieve aGVHD after allo-BMT.

PURPOSE: To determine the means, medians and reference intervals for TSH (thyroid-stimulating hormone) and fT4 (free thyroxine) for each month of gestation and for three trimesters in Korean pregnant women. METHODS: Serum samples were collected from 265 pregnant women with singleton gestation. Levels of TSH, fT4 were measured by immunoassay. After exclusion of subjects with positive antimicrosomal autoantibodies, the means, medians and reference intervals based on 2.5th and 97.5th percentiles for TSH, fT4 were determined. RESULTS: The study population consisted of 94 women in first trimester, 49 women in second trimester, and 122 women in third trimester. The trimester-specific reference intervals were: TSH (1st trimester: 0.03~2.72, 2nd: 0.27~2.29, and 3rd: 0.03~2.88 mIU/L), fT4 (1st trimester 4.50~19.75, 2nd: 4.70~12.98 and 3rd: 5.07~11.84 pg/mL). fT4 levels were significantly lower in the second and third trimesters. TSH levels were lower in the first trimester than second and third trimester, with gradual elevation in the second and third trimester. CONCLUSION: Levels of TSH, fT4 during pregnancy differ from those in non-pregnant women. Gestational age specific reference intervals will play a cental role in screening and diagnosis of thyroid disorders. Further studies for normal reference ranges during pregnancy are needed to create reference intervals in Korean pregnant women.
Autoantibodies ; Female ; Gestational Age ; Humans ; Immunoassay ; Mass Screening ; Pregnancy ; Pregnancy Trimester, First ; Pregnancy Trimester, Second ; Pregnancy Trimester, Third ; Pregnant Women ; Reference Values ; Thyroid Function Tests ; Thyroid Gland ; Thyrotropin

Objective:To conduct a systematic study of the immunologic response of rats to transplanted glutaraldehyde ( GA)-treated porcine blood vessels in vivo.Methods: The experiment was divided into two groups:fresh group and glutaraldehyde-treated group.Twenty cases of fresh and glutaraldehyde-treated porcine pulmonary arteries were subcutaneously embedded in rats.We compared the changes using HE staining and immunohistochemistry.Results:HE staining showed that there were stronger expression on day 12 and day 30 in the fresh group than that in the glutaraldehyde group.There were similar results in morphology in CD68,C3,IgG.The results of integral optical density ( IOD) in immunohistochemistry showed that IOD started rising from day 4 and got the peak on day 12 or day 30 and or fell on day 60.Conclusion: Innate immunity played an important role in the research on xenogenic immunological rejection mechanism.The immunogenicity of glutaraldehyde-treated xenogenic blood vessels is lower than that in fresh blood vessels.However there is still immunogenicity in glutaraldehyde-treated xenogenic blood vessels.We will explore better ways to obviously weaken the rejection.

Objective:To study the composition and influencing factors of hospitalization expenses of patients with sequelae of ce-rebral infarction caused by the dominant diseases of traditional Chinese medicine(TCM),so as to provide references for the fine management within the hospital and the medical insurance department to improve the payment policy suitable for the characteristics of TCM.Method:A total of 1 261 cases with ICD code 169.3 in the sample of tertiary traditional Chinese medicine hospitals from 2020 to 2022 were collected.Single factor analysis,correlation analysis,multiple linear stepwise regression and other methods were used to analyze the composition and main influencing factors of hospitalization expenses of patients with cerebral infarction sequelae.Re-sults:The average length of hospital stay of patients with sequelae of cerebral infarction was 16.71 days per time,the average hospi-talization cost was 24 148.83 yuan per time,and the proportion of TCM treatment cost per time was 54.43％.The results of stepwise regression showed that the length of stay,the condition of admission and the complication of pulmonary infection had a significant im-pact on the hospitalization expenses(P＜0.05),and the length of stay had the greatest impact.Conclusion:It is suggested that the hospital should optimize and improve the clinical pathway of TCM dominant diseases and establish an effective dynamic management mechanism,and the medical insurance department should improve the adjustment mechanism of the auxiliary list of TCM dominant diseases under the disease scoring payment.

BACKGROUND: Primary focal hyperhidrosis is characterized by overactivity of the sympathetic nervous function, and this has been effectively treated with endoscopic thoracic sympathetic denervation (ESD). The imbalance of sympathetic and parasympathetic nervous system that's created by ESD may affect the heart, lung and other thoracic organs. We analyzed the heart rate and ECG changes after performing ESD at our hospital, and this is the first such study that has been conducted on this. MATERIAL AND METHOD: Of the 263 patients who underwent ESD between October 1996 and October 2006, 130 had ECG before and after ESD, and they were classified into 3 groups according to the level of ESD: Group I (n=40) patients underwent ESD at the 2nd rib (T2ESD), Group II (n=80) at the 3rd rib (T3ESD) and Group III (n=10) at the 4th rib (T4ESD). RESULT: There was no mortality or major morbidity. Heart rate (HR) was significantly decreased from 71.6+/-10.6/min to 66.8+/-10.2/min after ESD (p<0.01); however, the PR (from 148.6+/-21.2 msec to 152.8+/-20.5 msec) and QTc (from 399.2+/-15.4 msec to 404.0+/-15.1 msec) intervals were significantly increased after ESD in the patients who suffered with primary hyperhidrosis (p<0.01). According to the level of ESD, there were significant changes in the HR and QTc interval in group I (T2ESD), the HR and PR interval in group II and the QTc interval in Group III. CONCLUSION: There were significant changes in the heart rate and ECG findings after ESD. The thoracic sympathetic denervation of T2, T3 and T4 affected the electrical activity of the heart at the resting state.
Electrocardiography ; Heart ; Heart Rate ; Humans ; Hyperhidrosis ; Lung ; Parasympathetic Nervous System ; Ribs ; Sympathectomy

BACKGROUND: Connexin 43-mediated gap junctional communication plays an important role in atherosclerosis. Numerous studies have demonstrated a correlation between mitral valve annular calcification and atherosclerotic disease. However, the relevance of connexin 43 to mitral valve disease remains unclear. We hypothesized that the mechanism contributing to mitral valve disease is associated with alterations in cell-to-cell communication mediated by changes in Connexin 43 expression. MATERIAL AND METHOD: Twenty male New Zealand rabbits were divided into two groups: animals in group 1 (n=10) were fed a normal chow diet, whilst those in group 2 (n=10) received a diet containing 1% cholesterol for 12 weeks. After sacrificing the animals, the mitral valves were excised and analyzed with immunohistochemical staining and Real-time Reverse Transcriptase polymerase chain reaction (real time RT-PCR). RESULT: Myofibroblasts and macrophages were found concentrated within the endothelial layer on the ventricular side of the leaflet in the cholesterol diet group. Immunohistochemial staining showed elevated expression of connexin43 in the cholesterol diet group. Real-time RT-PCR revealed increased connexin43 mRNA levels in mitral valves from hypercholesterolemic animals. CONCLUSION: Our finding that connexin43 expression is increased in mitral valves of hypercholesterolemic rabbits suggests that alterations in cell-to-cell communication via connexin43 containing gap junctions play a role in the development of mitral valve disease in hypercholesterolemia.
Animals ; Atherosclerosis ; Cholesterol ; Connexin 43 ; Diet ; Gap Junctions ; Humans ; Hypercholesterolemia ; Macrophages ; Male ; Mitral Valve ; Myofibroblasts ; Rabbits ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger ; Up-Regulation

OBJECTIVETo develop a near-infrared fluorescence imaging system based on the fluorescence properties of methylene blue.

METHODSAccording to the optical properties of methylene blue, we used a custom-made specific LED light source and an interference filter, a CCD camera and other relevant components to construct the near-infrared fluorescence imaging system. We tested the signal-to-background ratio (SBR) of this imaging system for detecting methylene blue under different experimental conditions and analyzed the SBR in urine samples collected from 15 Wistar rats with intravenous injection of methylene blue at the doses of 0, 1.4, 1.6, 1.8, or 2.0 0 mg/kg methylene blue.

RESULTSThe SBR of this imaging system for detecting methylene blue was affected by the concentration of methylene blue and the distance from the sample (P<0.05). In the urine samples from Wistar rats, the SBR varied with the the injection dose, and the rats injected with 1.6 mg/kg methylene blue showed the highest SBR (8.71∓0.20) in the urine (P<0.05).

CONCLUSIONThis near-infrared fluorescence imaging system is useful for fluorescence detection of methylene blue and can be used for real-time recognition of ureters during abdominal surgery.