AIM To express recombinant human cytochrome P450 3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18, and to employ them for in vitro metabolism studies of CYP3A4. METHODS Use Bac-to-Bac baculovirus expression system to recombinant baculovirus carrying cDNA of CYP3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18. Spodoptera frugiperda 9 (Sf9), cells were co-infected by recombinant viruses of CYP3A4 mutants, human NADPH-P450 oxidoreductase and cytochrome b5 to obtain recombinant proteins CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18 with metabolic activity. RESULTS The mRNA transcription of CYP3A4 mutants in Sf9 cells were validated by RT-PCR. Testosterone and 7-benzyloxy-4-(trifluoromethyl) coumarin were metabolized by the lysates of Sf9 cells infected by the recombinant viruses. CONCLUSION CYP3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18 with metabolic activity were successfully expressed by baculovirus-insect cell expression system. The results indicated that recombinant CYP3A4. 5 showed lower activity comparing to the wild type protein towards testosterone, while CYP3A4. 18 with higher activity, and for CYP3A4.3 and CYP3A4.4 showing similar activity to the wild type protein.

Objective To observe and analyze the outcomes o f open reduction and Y plate internal fixation in treatment of intra-articular calcaneal fractures.Methods Open reduction and Y plate internal f ix-ation by the extended L-shaped lateral approach were performed in 68pati ents with 82cases of intra-articula r calcaneal fractures under X-ray.Results82cases were followed up for an avera ge of 26months,and the clinical results were evaluated wit h the Maryland Foot Score.58cases ac hieved excellent results,16good,6fair,with the rate of excellent and good re sults being 90.24%.Conclusion Open reduction and Y plate internal fixation can obtain good clinical re sults in treatment of intra-articul ar calcaneal fractures.[

Objective: To investigate the regulation of respiratory syncytial virus CTL epitope fused with G protein antigen fragment G1 to the specific immunoresponses. Methods: The recombinant plasmid pET-DsbA-G1 or pET-DsbA-G1F/M2 was transferred into E.coli BL21(DE3) and the fusion protein DsbA-G1F/M2 or DsbA-G1 was expressed.The expressing product was induced and purified by affinity chromatography. The two proteins were used to immunized BALB/c mice i.p, respectively. Serum and spleen cells were collected regularly. RSV-specific CTL responses were measured by MTT, IgG and IgG1 and IgG2a antibodies by ELISA, neutralizing antibodies by plaque reduction assay. Results: The recombinant proteins were expressed successfully and the purity was over 90% after purified by affinity chromatography. The protein DsbA-G1F/M2 induced significant RSV-specific CTL responses, while DsbA-G1 without CTL epitope did not induce detctable CTL responses. Strong IgG antibody responses were elicited,indicated by both. IgG1 and IgG2a titers induced by DsbA-G1F/M2, while only IgG1 was induced by DsbA-G1.The ratio of IgG1/IgG2a was downregulated significantly. Both antigens induced high level of neutralizing antibodies, but the latter was a little lower. Conclusion: DsbA-G1F/M2, the fusion protein of a CTL epitope and G protein fragment G1 can induce both cellular immunity and humoral immunity. The activation of CTLs downregulates the ratio of IgG1/IgG2a.The more balanced immunoresponse is advantageous for improving safety of the candidate vaccine.

Objective To observe the glucose profile in type 2 diabetic gastroparesis.Methods 31 patients diagnosed with type 2 diabetes were enrolled into this study for measurement of gastric emptying of solids and continuous glucose monitoring to observe blood glucose 1evels for 72 hours on a balanced diet;the results were compared with 7 subjects with norinal glucose regulation.Results 58.1%of 31 type 2 diabetic patients were found to have delayed gastric emptying of solids.The average glucose level was lower after breakfast[(7.82±1.42)mmol/L vs(9.35±2.28)mmol/L,P<0.01]in the patients with gastroparesis than in those without.Maximal blood glucose level after breakfast[(10.21±2.17)mmol/L vs (12.24±2.82)mmol/L,P<0.01]was lower in the patients with gastroparesis but it reached the peak at a similar time.Two hour AUC was also lower after breakfast in the patients with gastroparesis[(877.62±Post-prandial glucose levelin type 2 diabetic patients with gastroparesis tends to be lower than those without.

Objective To construct the recombinant plasmid of fusion protein containing respiratory syncytial virus (RSV) cytotoxicity T lymphocyte (CTL) epitope M2:81-95 and heat shock protein (HSP) 70L1, and to investigate its immunogenicity after prokaryotic expression. Methods HSP70L1 gone was cloned from SMMC7721 cells. The M2:81-95 gene fragment was amplified by polymerase chain reaction (PCR) method. Plasmid pET-HSP70L1-M2 : 81-95 (pET-HSP70L1-M2) was constructed, identified and transferred into E. coli BL21 (DE3). Expression of HSP70L1-M2 : 81-95(HSP70L1-M2) was induced by isopropy-β-D-thiogalaetosidc (IPTG). The expressed protein was purified by affinity chromatography and renatured by gradient dialysis. The BALB/c mice were immunized with this fusion protein. IgG antibodies and the subtypes were detected by enzyme-linked immunosorbent assay (ELISA), and CTL responses were determined by methyl thiazolyl tetrazolium (MTT). Results The recombinant plasmid pET-HSP70L1-M2 was successfully constructed. The fusion protein HSP70L1-M2 was expressed in E. coll. The purified protein induced strong RSV-and CTL epitope-specific CTL responses and high titer of protein specific lgG antibody 4.87±0.35. The subtypes were IgG1 (5.53±0.28) and lgG2a (4.40±0.21) and IgG1/ IgG2a ratio was balanced. The titers of lgG, IgG1 and IgG2a in PBS control group were 0.33±0.17, 0.51±0.21 and 0, respectively, which werc significantly lower than those in immunized group (t = 3.512, 3.681, 5.856; P<0.05). Conclusions The recombinant plasmid pET-HSP70L1-M2 is successfully constructed and the fusion protein is expressed and purified. HSP70L1-M2 induced strong RSV-and CTL epitope-specific CTL responses and mixed T helper cell (Th)1/Th2 response in BALB/c mice.

BACKGROUND:Ligustrazine can effectively prevent and treat peritoneal adhesion and adhesive ileus after surgical treatment. OBJECTIVE:To prepare Ligustrazine nanoparticle spray which is prepared to achieve sustained release and improve clinical efficacy and to explore its optimal dose for preventing experimental peritoneal adhesions in rats. METHODS:Eighty rats were randomly divided into five groups:model group, sodium hyaluronate group, high-, medium-and low-dose Ligustrazine nanoparticle groups, with 16 rats in each group. After peritoneal adhesion model was established with rasp method, model group was immediately subject to abdomen-closing, while sodium hyaluronate group and Ligustrazine nanoparticle groups received sodium hyaluronate smearing and Ligustrazine nanoparticle spray (2.5, 5, 10 mg/kg), respectively. Al rats were kil ed at 1 and 2 weeks after modeling, to observe adhesions. The adhesion score was recorded. The adhesive tissue sections were stained with hematoxylin-eosin for pathological changes. The level of transformation growth factor-β1 in peritoneal fluid of rats was detected with ELISA assay. RESULTS AND CONCLUSION:Ligustrazine Nanoparticle spray had a sustained release effect, and prolonged the duration of action of drugs, thus achieving a better anti-adhesion effect post-surgery. The medium-and high-dose Ligustrazine nanoparticle sprays exhibited better anti-adhesion effects, and improved the rise of transformation growth factor-β1 level in the peritoneal fluid. As the drug concentration is low, the intraperitoneal administration in a spray manner is preferred. Because the total dose is limited, we define the optimal effective dose as 5 mg/kg.

ObjectiveTo observe the effect ofZuogui Jiangtang Jieyu Formula on the expressions of glutamate, NR2A and NR2B in hippocampus of diabetic rats with depression;To explore the mechanism of protective effect. Methods Diabetes with depression rat models were established and then were randomly divided into the model group, positive medicine group, high-, medium- and low-doseZuogui Jiangtang Jieyu Formula groups. Normal rats acted as normal group, 16 rats per group. After 28 days of administration, Open-field test was used to detect the behavior of the rats;glutamate content of hippocampus was detected by ELISA;the expressions of NR2A and NR2B in rat hippocampus were detected by immunofluorescence.Results Compared with normal group, automatic activity times of rats in model group decreased significantly (P<0.01);both glutamate content (P<0.01) and expressions of NR2A, NR2B (P<0.01) increased significantly. Compared with the model group, automatic activity times of rats in positive medicine group and high-doseZuogui Jiangtang Jieyu Formula group significantly increased (P<0.01);glutamate content dropped (P<0.01);expressions of NR2A and NR2B decreased (P<0.05).ConclusionZuogui Jiangtang Jieyu Formula can improve depressive behavior of diabetic rats with depression, which may be related to the regulation of glutamate content and expressions of NR2A and NR2B in hippocampus.

Objective To observe the clinical curative effect of ginseng cream stickon in patients suffering from lack of lactation after parturition .Methods Ninety patients suffering from lack of lactation after parturition were divided into three groups :the treatment group (patients with ginseng cream stick by acupoint application ) ,and 2 control groups (control with lactation elixir ,and control with decavitamin) .The treatment last for 5 days for each group .And the breast filling degree ,the lactigenous volume ,the neonatal weight ,artificial feeding times ,the artificial feeding volume and the prolactin level before and after the treatment were all recorded .Results Ginseng cream stick by acupoint application dramatically improved breast filling ,the lactigenous volume ,the neo‐natal weight ,artificial feeding times ,the artificial feeding volume and the neonatal urination frequency ,there was a significant differ‐ence between the treatment group and the two control groups (P<0 .05) .Conclusion Ginseng cream stick by acupoint application could significantly relieve lack of lactation ,meet the need of breastfeeding ,and increase the breast‐feeding rate .

Objective To study and establish the extraction technology of hypoglycemic components of total flavonoids and polysaccharides from Gynura divaricata (L .) DC ..Methods Based on the singer factor test ,the influences of extracting times ,diameter of drug powder ,soaking time ,solid-liquid ratio ,ethanol concentration ,and extracting time on extraction ratio of total flavonoids were studied .The best extraction technology of total flavonoids from Gynura divaricata (L .) DC .was es-tablished through L9 (3)4 orthogonal tests .On the base of previous studies combine with L9 (3)4 orthogonal tests ,the best ex-traction technology of total polysaccharides from Gynura divaricata (L .) DC .was established .Results The optimized extrac-tion technology of total flavonoids was confirmed as follows :drug powder of less than 10 mesh diameter were soaking 0 .5 hour with 80% ethanol ,and solid-liquid ratio 1∶20 ,and extracting twice ,each time one hour .The optimized extraction technology of total polysaccharides was confirmed as follows :add 20 times water to the residue of ethanol ,extraction temperature 90℃ , and extracting twice ,each time one hour .The extraction rate of both total flavonoids and polysaccharides were more than 80% .Conclusion The extraction technology of hypoglycemic components of total flavonoids and polysaccharides from Gynura divaricata (L .) DC .established in this study is stable ,reproducible ,and lays a foundation for the further studies on hypogly-cemic components in Gynura divaricata (L .) DC .

Objective:To investigate the serum measles antibody in children with tumor and to provide the clinical evidence for measles vaccination strategy for this special population.Methods:From January 2016 to December 2018, the blood samples of children who were diagnosed with hematological malignancy or solid tumors and received chemotherapy in the Department of Hematology or Oncology Surgery of Children′s Hospital of Fudan University were collected. Enzyme-linked immunosorbent assay was used to quantitatively detect the level of measles IgG antibody, and dynamically monitor the changes of measles antibody level during chemotherapy. Kruskal-Wallis test and chi-square test were used for statistical analysis.Results:A total of 441 children with tumors were enrolled, with the positive rate of measles antibody of 79.1%(349/441), and only 43.3%(191/441) of children had the protective level of IgG antibody. There was a statistically significant difference of the antibody protection rate in children aged0.05). The protection rate of serum measles antibody in children with hematological malignancy and solid tumor were 45.6%(78/171) and 41.9%(113/270), respectively, and there was no statistically significant ( P>0.05). There were 16.3%(16/98) of children who were observed to lose the pre-existing protective antibody during chemotherapy. There was no statistically significant difference of the protection rate of serum among children who had finished chemotherapy 0.05). Conclusions:Serum measles antibody is below the protective level in more than 50% of children with malignancy after chemotherapy. Chemotherapy can compromise the protective antibody against measles. It is recommended for this special population to re-schedule measles vaccine after individualized evaluation to acquire the immuneprotection against measles.