Chronic hepatitis B (CHB) is still a global public health problem, resulting in the high risk of decompensated cirrhosis and hepatocellular carcinoma.At present, there are no single available medications that can induce both potent HBV DNA suppression, and high rates of HBeAg and HBsAg clearance.Therefore, there is great interest in developing combination therapies for patients with chronic HBV infection, which can achieve sustained viral suppression and HBsAg negative inversion after a finite course of treatment leading to clinical cure.This article reviews the current status and advances of combination therapy for CHB.

Objective To investigate the expression and purification I278T-mutant human cystathionineβsynthase(CBS) in E . coli .Methods Site-directed mutagenesis by overlap extension using the polymerase chain reaction (PCR) was employed to construct mutant plasmids pGEX4T-1-CBS(I278T) ,which was induced and expressed in a medium containing 3% ethanol ,purified by affinity chromatography to obtain mutated CBS (I278T) protein .The activity ,UV-visible absorption spectroscopy ,protein particle size and Zeta potential of the purified protein were measured .Results Plasmid pGEX4T-1-CBS(I278T) was successfully constructed .The yield ,the specific activity and activity recovery of purified mutant CBS (I278T ) protein were 2 .3 mg/L ,21 .4 U/mg and 22 .6% .S-adenosylmethionine(AdoMet) with final concentration of 1 mmol/L showed no activation toward mutant CBS (I278T) protein .Ac-cording to UV-visible absorption spectroscopy analysis ,purified mutant CBS(I278T) had characteristic absorption peaks at 429 nm and 550 nm for heme-binding proteins .Protein average particle size was 7 .5 -10 .1 nm ,mainly in the form of tetramers ,and Zeta potential was - 16 .3 mV .Conclusion The methods of expression ,purification and identification of I278T-mutant human cystathionineβsynthase in E .coli were successfully established .

BACKGROUND:The establishment of a good blood supply is a key mechanism for successful implantation of engineered tissues. OBJECTIVE:To observe the effect of basic fibroblast growth factor on the migration of human adipose-derived stem cells via implanting the human adipose-derived stem cells and sodium hyaluronate composite graft at the subcutaneous site of BALB/C mice, in order to explore an optimal scheme for soft tissue reconstruction. METHODS:Human adipose-derived stem cells were isolated from the adipose tissue of healthy cosmetic patients which received liposuction, and the cells were subcultured. Then 5×109/L passage 3 cellsuspension labeled by cm-dil was prepared. The working solution containing 2 mg/L basic fibroblast growth factor was prepared. Composite tissue al-lografts which were the mixtures of 0.25 mL sodium hyaluronate, 0.2 mL cellsuspension and 0.05 mL working solution or DMEM were implanted into the subcutaneous site of both sides of the mouse back. Specimens were taken at 6 weeks after operation and were evaluated histological y after hematoxylin-eosin and vascular immunofluorescent staining. RESULTS AND CONCLUSION:No necrosis, liquefaction, nodular tissue or gel remained in operated position. The hematoxylin-eosin staining showed the main components of the specimens were the adipose tissue and the loose connective tissue. The immunofluorescence staining showed the overlaps between the cm-dil fluorescence from human adipose-derived stem cells and the FITC fluorescence from the vascular endothelium in the experimental group were more than those in the control group (P<0.05). Basic fibroblast growth factor promotes the migration and the differentiation of human adipose-derived stem cells in the sodium hyaluronate scaffold into vascular endothelium.

BACKGROUND:Expanded polytetrafluoroethylene is a kind of porous polymer materials which is commonly used as clinical implants, and it has good biocompatibility, and is not easy to deformation or metamorphism. There is no existence of inflammation absorption reaction, and it al ows the cel migration and tissue ingrowth.
OBJECTIVE:To study the biocompatibility of expanded polytetrafluoroethylene scaffold and human adipose-derived stem cel s.
METHODS:The passage 4 human adipose-derived stem cel s were co-cultured with expanded polytetrafluoroethylene scaffold in vitro. The morphology and function of cel s adhered to the scaffold were observed by inverted phase contrast microscope, and cel adhesive rates and proliferation rates were also calculated by MTT assay.
RESULTS AND CONCLUSION:The inoculated cel s were round and bright, distributed on the surface of scaffolds uniformly, with good cel viability. After 3 hours a large number of adherent cel s were observed from the micrograph;after 24 hours there were a smal amount of short-spindle adipose-derived stem cel s. After cultured for 3 days, the short fusiform or polygon cel s could be seen clearly. After cultured for 7 days, the number of cel s increased significantly, few cel s fel off from the scaffold, and cel adhesion rate was up to an average of 95.7%. Meanwhile, the cel s revealed normal splitting proliferation rate. These findings indicate that human adipose-derived stem cel s are able to attach, grow and proliferate wel on the scaffold. Expanded polytetrafluoroethylene reveals excel ent cel ular compatibility and can be used as a vehicle for adipose tissue engineering.

Objective To establish a mouse model of kidney-yin and kidney?yang deficiency after oral administra?tion of hydrocortisone, and to explore the related evaluation factors. Methods The model was established by oral adminis?tration of hydrocortisone to induce kidney?yin and kidney?yang deficiency in mice. The survival and body weight of the mice were observed. The serum content of adrenal cortical hormone ( ACTH) , cortisol ( Cor. ) in the hypothalamic?pituitary?ad?renal (HPA) axis, and thyroid stimulating hormone (TSH), triiodothyronine (T3), thyrox (T4) in the hypothalamic?pi?tuitary?thyroid (HPT) axis, follicle?stimulating hormone (FSH), estradiol (E2), testosterone (T) in the hypothalamic?pituitary?gonadal ( HPG) axis were determined by radioimmunoassay. Results The body weight of kidney?yin and kidney?yang mice were decreased, the serum ACTH, Cor, TSH, T3, T4 contents were decreased, the serum FSH, E2, T contents were increased in the kidney?yang deficiency model mce ( P<0. 01 ) , and those parameters in the kidney?yin deficiency model mice were changed in opposite direction. Conclusions It is found that the hormone levels of ACTH, Cor, TSH, T3, T4, FSH, E2 and T in kidney deficiency mice are changed, and cortisol can be used as an important index to evaluate the model of kidney deficiency induced by glucocorticoid.

Objective To observe the effects ofLiuwei Dihuang Decoction on cAMP and PDE3B in adipose tissues of rats with type 2 diabetes;To explore its mechanism.MethodsTotally 80 SD rats were randomly divided into two groups:control group and molding group. Rats in the molding group established the type 2 diabetic models by feeding high sugar and fat diet combined with intraperitoneal injection of Streptozotocin. The successful modeling rats were divided into model group, Rosiglitazone group, andLiuwei Dihuang Decoction group. Administration groups were given relevant medicine for gavage, while model group and blank group were given the same amount of normal saline for 30 days. FBG levels were detected, and cAMP and INS levels were detected by ELISA. The protein expressions of PDE3B in adipose tissues were determined by Western blot. The mRNA expressions of PDE3B in adipose tissues were detected by RT-PCR.Results Compared with the model group, the FBG and INS levels in type 2 diabetic rats were reduced in Rosiglitazone group andLiuwei DihuangDecoction group (P<0.05,P<0.01). InLiuwei Dihuang Decoction group, the protein and mRNA expressions of PDE3B in the adipose tissues were higher, and cAMP levels were lower than the model group (P<0.01).ConclusionLiuwei Dihuang Decoction can increase PDE3B expression, and decrease cAMP level, which may be one of the mechanisms for improving insulin resistance in type 2 diabetic rats.

Objective To study the effect of the exogenous recombinant human FGF-basic with different concentrations upon inducing human adipose-derived stem cells (hASCs) to differentiate into adipose cells,and the optimum concentration of exogenous rh-bFGF by experimental research.Methods hASCs were isolated and extracted by enzymatic digestion from the liposuction aspirate.hASCs using adipogenic supplement were divided into experimental group and blank group:the experimental group of adipogenic supplement was divided into adding the exogenous rh-bFGF 10 ng/ml,20 ng/ml and 40 ng/ml,the blank group of adipogenic supplement was cultured without exogenous rh-bFGF.MTT method was used to detect the adipocytes proliferation.The oil red O staining was used in the qualitative analysis on the time of newly forming adipocyte cells.Western blot was used to detect the effects of rh-bFGF on the expression of lipid droplets surface protein CIDEC at different stages during the culture.Results The experimental group could obviously shorten the period of inducing hASCs to differentiate into adioicytes,and promote the proliferation of adipocytes.The formation rate and the proliferation of adipocytes in the group adding 40 ng/ml rh-bFGF were superior to those in the experimental group else and blank group.The average time of the newly formed lipid droplets by adding 40 ng/ml rh-bFGFwas (11.5±1.9)h.The average absorbance of cell proliferation by adding 40ml rh-bFGF was 0.52 ±0.10.The CIDEC expression quantity of adding 40 ng/ml rh-bFGF group was also superior to that in the experimental group and blank group.Conclusions rh-bFGF in hASCs adipogenic supplement could promote the proliferation of adipocytes and dramatically accelerates the program of hASCs differentiating to adipocytes,in which the optimum concentration of rh-bFGF is 40ng/ml.

Objective The evaluation management of scientific research projects is the core work content of scientific research management institutions at all levels.The primary causes of bias in project evaluation are the acquaintance social network relationship and the unit-based unitism,especially in the case of small academic circles.The purpose of this essay is to study how to circumvent this problem,to select projects objectively and fairly,and to benefit the evaluation and management of scientific research projects at all levels.Methods Including information construction,expert database construction,expert rotation system,expert avoidance system,supervision and examination system,peer-expert review and strengthen the expert project evaluation.For the first time,we proposed a double-blind,random,centralized,and group-based independent evaluation system.Results This method has a positiveeffect on view.Conclusions In the case where the relative or absolute numberof experts in a single field is small,implementing a series of normative systems and measures through organizational management can avoid interference factors that affect the project evaluation effectively and achieve more scientific and fair results.

Objective To evaluate the clinical efficacy of interferon(IFN) α-2b,adefovir dipivoxil (ADV),granulocyte-macrophage colony stimulating factor (GM-CSF) and hepatitis B vaccine in the treatment of patients with HBeAg-positive chronic hepatitis B (CHB).Methods Two hundredand forty HBeAg-positive CHB patients admitted in the First Affiliated Hospital,Zhejiang University School of Medicine during November 2013 and December 2015 were retrospectively reviewed.They were randomly assigned to four groups with 60 cases in each group,to receive IFNα-2b (group A),IFNα-2b + ADV (group B),IFNα-2b + ADV + GM-CSF (group C) or IFNoα-2b + ADV + GM-CSF + hepatitis B vaccine (group D) for treatment,respectively.All patients were treated for 48 wks and then followed up for 24 wks.The HBsAg serological response,HBeAg serological response,virological response,biochemical response,histological response and adverse effects were compared among 4 groups.Results After 48-wk treatment,the HBsAg negative conversion rate of group D was significantly higher than that of group A and B (x2 =8.634 and 8.634,both P ＜ 0.01);the seroconversion rate of HBsAg in group D was significantly higher than that in group A and group B (x2 =7.149 and 7.149,both P ＜0.01).The baseline drop rate of HBsAg in group C and D was higher than that in group A (t =4.194 and 4.508,both P ＜0.01).In 24-wk follow-up,HBsAg negative conversion rate was as same as that after 48-wk treatment;the seroconversion rate of HBsAg in group D was significantly higher than that in group A and B (x2 =8.634 and 8.634,both P ＜0.01).The baseline drop rate of HBsAg in group C and group D was higher than that in group A (t =4.546 and 4.969,both P ＜0.01).After 48-wk treatment,the HBeAg negative conversion rate in group D was significantly higher than that in group A (x2 =8.792,P ＜ 0.01);the HBeAg seroconversion rate of group C and D was higher than that of group A (x2 =7.064 and 10.159,P ＜0.01).In 24-wk follow-up,the HBeAg negative conversion rate in group C and D was higher than that in group A (x2 =10.159 and 13.713,P ＜0.01);the HBeAg negative conversion rate in group D was higher than that in group B (x2 =8.155,P ＜0.01);the seroconversion rate of HBeAg in group C and D was higher than that in group A (x2 =10.506 and 12.857,P ＜ 0.01).After 48-wk treatment,the negative rate of HBV DNA in group B,C and D was significantly higher than that in group A (x2 =12.452,17.062 and 20.670,all P ＜0.01).In the 24-wk follow-up,the negative rate of HBV DNA in group B,C and D was also significantly higher than that in group A (x2 =21.121,26.880 and 33.611,all P ＜ 0.01).After 48-wk treatment,the alanine aminotransferase (ALT) normalization rate of the group of C and D was significantly higher than that in group A (x2 =8.711 and 8.711,both P ＜0.01).In the 24-wk follow-up,the ALT normalization rate in group C and D was significantly higher than that in group A (x2 =8.076 and 9.624,P ＜0.01).After 48-wk treatment,the improvement rate of inflammation or fibrosis in the group of A,B and C was significantly higher than that in the group A (x2 =8.543,13.348 and 16.205,all P ＜ 0.01).There were no half-way withdrawal and no serious adverse reactions.The main adverse reactions were fever,headache and fatigue,and no significant difference of above reaction was observed among 4 groups (P ＞ 0.05).Conclusion The new anti-virus/immunoregulation therapy containing IFNα-2b + ADV + GM-CSF + hepatitis B vaccine has a better therapeutic effect for patients with HBeAg-positive CHB.

Objective:To investigate the changes of liver pathology and its influencing factors in patients with chronic hepatitis B viral (HBV) infection and low level of serum alanine aminotransferase (ALT).Methods:The clinical data of 135 with chronic HBV infection patients, in whom the serum ALT levels were less than two times of the upper limit of normal (ULN), were collected from the Affiliated Hospital of Medical School of Ningbo University and the First Affiliated Hospital, Zhejiang University School of Medicine during July 2017 and July 2019. The result of hepatic histological examination was reviewed, and the risk factors of obvious liver inflammation (G≥2) or fibrosis (S≥2) in patients were analyzed with Logistic regression analysis.Results:The pathological examination of liver tissue revealed G≥2 or S≥2 in 52 cases (38.5%). The univariate analysis showed that age, family history of HBV infection, ALT 1-<2×ULN, aspartate aminotransferase(AST)≥1×ULN, low platelet count(PLT)and prolongation of prothrombin time(PT)were associated with G≥2 or S≥2 in chronic HBV infection patients with low level ALT ( P<0.05 or <0.01). Multivariate Logistic regression analysis showed that age( OR=1.052, 95% CI 1.007-1.100), family history of HBV infection( OR=5.448, 95% CI 2.191-13.548)and AST( OR=1.042, 95% CI 1.005-1.081)were independent risk factors of G≥2 or S≥2 in chronic HBV infection patients with low level ALT ( P<0.05 or <0.01). Conclusion:Age, family history of HBV infection and AST level can be used to judge the severity of liver pathological changes and necessity of antiviral treatment for patients with chronic HBV infection having ALT<2×ULN.