Objective To rapidly construct hepatitis B virus(HBV)adefovir(ADV)‐resistant strain(RT A181T)infectious clone by using SOE‐PCR ,to observe the expression of recombinant plasmids in Huh7 cells and to establish the in vitro cell model of HBV ADV‐resistant strain(RT A181T) .Methods The conservative primers were designed ,the full‐length HBV genome was amplified from serum in the patients with ADV‐resistant chronic hepatitis B .Then ,the 1 .3‐fold genome‐length infectious clone pHBV1 .3 was constructed by using the SOE‐PCR technique ,which was transfected into human hepatoma cells Huh7 cell line .ELISA ,Western Blot and Real‐time PCR were adopted to detect infectious cloning replication and expression ,meanwhile the antiviral drugs lamivu‐dine(LAM ) and ADV were used to verify the infectious clone copy and inhibition of expression .Results The HBV ADV‐resistant infectious cloning plasmids pHBV1 .3 was successfully constructed .This plasmid could be effectively replicated ,transcripted and ex‐pressed in Huh7 cell lines .LAM could effectively inhibit the replication and expression of the infectious clone ,while ADV could not inhibit the replication and expression of the infectious clone .Conclusion The constructed infectious clone plasmids pHBV1 .3(RT A181T ) of HBV ADV‐resistant strain can efficiently replicate and express protein in vitro ,its transfected cells can be used in the study of HBV replication mechanism and antiviral study .

Objective: To investigate the effects of hepatitis B virus (HBV) X protein (HBx) on the expression of tumor necrosis factor-α (TNF-α) in glomerular mesangial cells (GMCs) and the underlying intracellular signal pathways. Methods: The plasmid pCI-neo-X that carries the X gene of hepatitis B virus was transfected into cultured GMCs. HBx expression in the transfected GMCs was assessed by Western-blot. TNF-α protein and mRNA were assessed by ELISA and semi-quantitative RT-PCR, respectively. Three kinase inhibitors-U0126, an inhibitor of extracellular signal-regulated kinases (ERKs);lactacystin, an inhibitor of nuclear factor-κB (NF-κB);and SB203580, a selective inhibitor of p38 MAP kinase (p38 MAPK) were used to determine which intracellular signal pathways may underlie the action of HBx on TNF-αexpression in transfected GMCs. Results:A significant increase in HBx expression in pCI-neo-X transfected GMCs was detected at 36 h and 48 h, which was not affected by any of those kinase inhibitors mentioned above. A similar increase in the expression of both TNF-αprotein and mRNA was also observed at 36 h and 48 h, which was significantly decreased in the presence of U0126 or lactacytin, but not SB203580. Conclusions:HBx upregulates TNF-αexpression in cultured GMCs, possibly through ERKs and NF-κB pathway, but not p38 MAPK pathway.

Objective To observe the effects of silencing IGF-1 receptor on the expression of Caspase12 , a endoplasmic reticulum specificity apoptosis protein of the peroxidation damage in L02 cells. Methods Four the recombined plasmids expressing short hairpin RNA (shRNA) specific for sileincing IGF-1R gene were constructed and these shRNAs were potentially targeted on different mRNA sequences of the IGF-1 receptor gene (Sh-H-IG1R- 1108, Sh-H-IGF1R-2797, Sh-H-IGF1R-3215 and Sh-H-IGF1R-3787). These constructs were classed as the experimental group. Another two constructs containing an irrespective sequence and no sequence were defined as a positive control group a negative control group , respectively. All these constructs were transfected into the L02 cells by Lipefectamine 2000. The interference effect was detected by RT-PCR after 24 h and the plasmid stably silencing the expression of IGF-1 receptor was selected for further analysis which was divided into four groups: the blank control group(Group A), the peroxidation damage group(Group B), the negative plasmid transfection group (Group C) and the positive plasmid transfection group (Group D). The expression of Caspase12 was detected by Westen Blot after 12 h. Results The amplification folds of experimental groups were statistically significantly lower than those of the Sh-H-IGF1R-V1 group and Sh-H-IGF1R-V2 group (P < 0.05). The silence effect of the Sh-H-IGF1R-3215 group (0.02 ± 0.01) and the Sh-H-IGF1R-3787 group (0.05 ± 0.05) were most stable and the repeatability of the later was better. The expressions of Caspase12 in Group B (2.41 ± 0.03), Group C (2.45 ± 0.02) and Group D (3.60 ± 0.06) were higher than Group A (0.34 ± 0.02) (P < 0.01). The Group Cexpression was higher than that of the Group B (P > 0.05). The Caspase12 expression in Group D was higher than that in Group B and C (P < 0.01). Conclusion (1) Of all the four constructs in this study, Sh-H-IGF1R-3787 is the most suitable for silencing IGF-1 receptor. (2) Silencing IGF 1R expression using shRNA plasmids can aggravate the L02 cells apoptosis, suggesting that the IGF-1/IGF 1R signal pathway might be involved in regulating the activation of apoptosis in L02 cells with peroxidation damag via endoplasmic reticulum stress.

Objective To analyze the subject frequency and score proportion of the national unified examination papers of Nursing Research according to the topic content,in order to offer proposals for the reformation and optimization of the examination,and provide self-test-takers with references of improving learning methods and results.Methods The papers of the subject of Nursing Research during October 2008 to January 2013 had been statistically analyzed following the syllabus.Results Comprehensive coverage of knowledge point existed in the papers of the recent five years.The theme of measurement of research tools (5.8％) and basic procedures of nursing research (7.0％) took a smaller proportion compared with nursing research design (16.0％) and nursing papers writing(16.3％).Conclusions The contents of the 10 papers of the past 5 years concentrate high attentions and emphasis on the comprehension and application of the basic theory and methods,which accord with the goals and requirements of this subject in the training of the research ability.

Ischemic reperfusion is the common mechanism of some chronic diseases such as type 2 diabetes,coronary heart disease,hypertension,and so on.Previous studies of insulin resistance is mainly focused on the relationships of these chronics,while now,in recent years,the research between the insulin resistance and ischemic reperfusion has gradually become one of the new and hot points,closely related to the patient's prognosis.In this paper,we will review the insulin resistance and ischemic reperfusion according to the reported literature review.

It is urgent to construct the integrity of medical students on campus.Students of the first and fifth grade which are also the two special stages are investigated in the survey,According to the comparison of the honor credibility knowledge and behavior,the article summarized the effectiveness of contemporary medical education,and discussed the dialectical relationship between the integrity education of the medical students and medical integrity,to give full play to the clinical practice teaching as a significant role of the integrity education of medical students.

Infection is one of the most familiar complications in children with idiopathic nephrotic syndrome.Although vaccination is an effectual measure in prevention of secondary infections, it still faces many problems.On the one hand, children′s own immune status (immune system dysfunction, administration with immunosuppressants, etc.) lends additional uncertainties to whether the immune system can produce sufficient protective antibodies after vaccination.On the other hand, potential risks (recurrence of nephrotic syndrome and induction of vaccine related diseases) need to be comprehensively assessed at the time of vaccination.In this paper, the safety, effectiveness and timing of va-ccination in children with nephrotic syndrome in the post-COVID-19 period were summarized, and possible problems of vaccination against severe acute respiratory syndrome Coronavirus 2(SARS-CoV-2) were discussed.

Objective To investigate the correlation of postoperative cognitive dysfunction (POCD)and the preoperative he-moglobin (HbA1c)in elderly patients with type 2 diabetes mellitus (T2DM).Methods 82 patients with T2DM who were underwent non-cardiac surgery were included in this study,group A had 37 patients with POCD after surgery,group B had 45 patients with non POCD,the patients in two groups used the same anesthesia method,compared the general data and pre-operative HbA1c,fasting blood glucose levels of two groups,compared the incidence of POCD in patients with HbA1c in dif-ferent levels.Results There was no significant difference in gender composition,age,body mass index,preoperative MMSE score,hypertension rate,operation time and bleeding volume in operation,and there was no significant difference in fasting blood glucose of patients in two groups.Fasting blood-glucose (FBG)of two groups were 7.1±2.6 and 6.7±1.8 mmol/L (t=0.821,P=0.424),it was no significant difference;HbA1c of two groups were (7.2±1.3)% and (6.3±1.0)%,the group A was higher than the group B (t=3.543,P=0.001),there was significant difference.The incidence rate of POCD of the group AH was 58.1%(25/43),while the group AL was 30.8% (12/39),the group AH was higher the group AL(χ2=5.131,P=0.024),there was significant difference.Conclusion Preoperative determination of HbA1c in elderly patients with T2DM after surgery has forecast values for the occurrence of POCD,to control HbA1c in the low level can reduce the occurrence of POCD in elderly patients with T2DM.

Objective To observe the effects and safety of rapamycin (Rap) in combination with cyclosporine A (CsA) and steroid (Pred) as triple regimen for prevention of acute rejection after renal transplantation.Methods Fifty patients with de novo cadaveric kidney allograft were randomized into two groups. In the study group there were 30 patients treated with Rap, CsA, and Pred, while in the control group there were 20 patients with conventional triple therapy of azathioprine (Aza), CsA, and Pred. Same dosages of CsA and Pred were used in both groups. Any adverse event after treatment was observed and recorded in detail. The laboratory investigations were checked and analyzed periodically.Results The pre-protocol population included 26 patients in study group and 18 patients in control group. The 2-year patient and graft survival rates in both groups were 100 %. The incidence of acute rejection was 3.7 % (1/27) in study group, 25 % (5/20) in control group, respectively. All episodes of acute rejection were reversed by methylprednisolone. The trough levels of CsA kept same in both groups, but CsA dose in study group was lower than that in control group. Blood concentrations of total cholesterol and triglyceride in study group were higher than those in control group. Conclusion Rap is an effective and safe immunosuppressive drug for prevention of acute rejection after renal transplantation. Rap combined with CsA allows reducing the dose of CsA. But its main side effect is hyperlipidemia.

Objective That decline of estradiol in serum of postmenopausal women can result in dry eye has been accepted at present. However, the change of estradiol in tear of postmenopausal women has been not reported ever. This study was to discuss the relationship between dry eye syndrome and estradiol in tears and serum of postmenopausal women. Methods Total 62 eyes of 62 women were collected in this trial. The aim of this trial was informed orally and the informed consent was obtained from all the subjects. The 32 right eyes were from menopausal women for more one year and 30 right eyes were from premenopause women. The dry eye was diagnosed based on the abnormalities of tear break-up time (BUT), Schirmer I test, corneal fluorescence and subjective symptom of patients. The periphery blood specimen was collected from all the subjects, and tear specimen was obtained and quantified using filter paper. Estradiol contents in tears and serum were determined by chemiluminescence method. The bulbar conjunctiva was obtained by impression cytology, and the microstructure of conjunctiva was observed under the scanning electron microscopy. Results In comparison to the premenopause group, the content of estradiol in tears and serum in menopause group was significantly reduced(73. 50 ±24.47 vs 17. 43 ± 10. 92) (U = -6. 396, P =0. 001). In the premenopause group, the estradiol content between tear and serum showed the positive correlation (r = 0. 838, P = 0. 001) . However, no statistical correlation was found in estradiol content between tears and serum in menopause group(r =0. 127, P = 0. 487). The maicrostructure changes were seen in the dry eyes of postmenopausal women, and decrease of goblet cell and loosen of intercellular junction were found under the electron microscope. Conclusion The content of estradiol in tears in menopause group is significantly lower than that in the premenopause group. Estradiol content in tears and serum of the postmenopausal women showes insignificant correlation. This result indicates that the topical substitute therapy with estradiol is possible in dry eye.