Objective To assess the distribution of recombinant fusion protein dTMP-GH in mice and to determine whether it is of tar-geted distribution characteristics. Methods A laboratory scale preparation of dTMP-GH recombinant fusion protein was obtained. Protein dT-MP-GH was labeled with radioactive 125 I,then mice were sacrificed at 5 min,15 min,30 min,1 h,2 h,4 h,8 h,12 h,24 h after tail vein injec-tion of 125 I-dTMP-GH at a dose of 100 μg/kg,and the organs and tissues ( heart,liver,spleen,kidney,bone and thyroid) were collected for radioactive counting. Results Preparation of purified ( >98%) dTMP-G was obtained. 125 I labeling rate was 71. 53%,radiochemical purity was 96. 53%,and specific activity was 0. 22 MBq/μl. 30 min after tail vein injection of 125 I labeled dTMP-GH,radioactivity accounted for 10% of the total injected in femoral,and metabolism was carried via liver and kidney over time. Conclusion Fusion protein mainly distribu-ted in bone marrow via tail vein injection in mice,which expressed that dTMP-GH has the characteristics of selective distribution in bone mar-row tissue.

Objective To explore the changes of gut microbiota in response to abdominal and pelvic radiotherapy and its potential relationship with intestinal infection.Methods Irradiation was delivered to the abdominal region of BALB/c mice,following the regular human pelvic-radiotherapy protocol,2.0 Gy/d,continuous 5 d/week.Samples of ileum tissue and the intestinal content were collected at different time points of irradiation procedure,including after 3 and 5 weeks,and at 1 week after 6 weeks of irradiation.Quantitative RT-PCR was used to measure the mRNA level of antimicrobial peptides and pro-inflammtory factors.Bacterial translocation was determined by PCR.The gut microbiota was characterized by the denaturing gradient electrophoresis assay.Results The expressions of cryptdin-1 and cryptdin-4 were decreased after 3 weeks of irradiation and at 1 week after 6 weeks of irradiation(t =-7.43,-3.54,-4.72,-4.27,P ＜ 0.05),while they were significantly increased at the 5 weeks of radiation (t =6.15,5.75,P ＜ 0.05).The diversity index and richness of gut microbiota after 3 or 5 weeks irradiation were significantly decreased (t =-3.49,-4.19,-3.44,-4.97,P ＜ 0.05).The gut microbiota dysbiosis of the irradiated mice was characterized with the decrease of probiotics of Lactobacillus and the increasing of opportunistic pathogen of Escherichia coli,Shigella flexneri,et al.Bacterial translocation episodes were more frequently in the irradiated mice than that of control animal.The mRNA levels of IL-1β、IL-6 and TNF-α were significantly increased after 3 or 5 weeks of irradiation (t =4.85,6.16,7.71,4.60,4.86,5.97,P ＜ 0.05).Compared with the control,the expression levels of IL-1β and TNF-α at the 1 week after 6 weeks of irradiation ending was also obviously enhanced (t =3.67,5.88,P ＜0.05).Conclusions Pelvic radiotherapy can induce abnormality of enteric antimicrobial peptides and may result in gut microbiota dysbiosis.The disturbed gut microbial flora may further trigger an incurrence of bacterial translocation and enteritis.Therefore,the gut microbiota may be a potential interfering target to alleviate radiotherapy adverse effect.

Objective To investigate the effects of rTMP-GH, a recombinant fusion protein of thrombopoietin mimetic peptide (TMP) and human growth hormone (GH), on the proliferation and thrombocytopoiesis of cultured megakaryocytes. Methods After being treated with 100 ng/ml of rTMP-GH for 7 d, Dami cells, a kind of megakaryocyte cell line, were analyzed by observing the numbers of colony forming unit. Meanwhile, Western blotting and RT-PCR were applied to detect the expression changes of globin transcription factor-1 (GATA-1), which is a main regulator of thrombocytopoiesis in megakaryocytes. Results The numbers of colony forming unit were markedly increased in cultured Dami cells incubated with rTMP-GH. Compared with normal control and GH treatment groups, both the mRNA and protein levels of GATA-1 were up-regulated significantly in Dami cells treated with rTMP-GH. Conclusion rTMP-GH has a strong ability to promote the proliferation and thrombocytopoiesis of megakaryocytes.

Objective To fulfill high-efficient expression of rTMP-GH,a recombinant fusion protein of thrombopoietin mimetic peptide and human growth hormone,in Pichia pastoris.Methods cDNA fragment coding for rTMP-GH was amplified by PCR,and subsequently cloned into the expression vector pPIC9K.The linearlized plasmid pPIC9K-rTMP-GH was transformed into Pichia pastoris GS115 cells and screened in MD/MM plates under pressure of G418.The recombinant fusion protein rTMP-GH was identified by Western blotting,and its biological activity was analyzed by its ability on colony formation of megakaryoblasts.Results The pPIC9K-rTMP-GH expression plasmid was successfully constructed.GS115 cells with high expression of rTMP-GH was screened out,and the rough purified rTMP-GH showed promotive effect on megakaryoblast colony formation.Conclusion High-efficient expression of rTMP-GH in Pichia pastoris is achieved.

Objective To study the application value of neutrophil elastase (NE), fibrinogen (Fib) combined with tumor necrosis factor-α (TNF-α) in the prognosis prediction of severe pneumonia in children. Methods Eighty-two children with severe pneumonia who were admitted into the Yuhang District Maternal and Child Health Hospital of Hangzhou in Zhejiang Province from July 2016 to September 2018 were treated as a severe group, and the children with severe pneumonia were subdivided into a survival group (70 cases) and a death group (12 cases) according to the prognosis; another 90 children with common pneumonia who were treated in our hospital at the same time were selected as a general group; and 85 normal children who received physical examinations at the same time as a healthy control group. The levels of serum NE, Fib and TNF-α in the three groups were measured by enzyme-linked immunosorbent assay (ELISA), and the pneumonia severity index (PSI) was calculated in the severe group and the general group; Spearman correlation analysis was used to analyze the correlation between NE, Fib, TNF-α and PSI;the NE, Fib and TNF-α levels were evaluated to predict the prognosis of children with severe pulmonary disease;the receive operating characteristic (ROC) curve was drawn to evaluate the prognostic value of NE, Fib, TNF-α in children with severe pulmonary disease. Results The expression levels of serum NE, Fib and TNF-α in the severe group were higher than those in the general group and the healthy control group [NE (μg/L): 127.5±12.3 vs. 75.1±6.6, 24.3±5.9, Fib (g/L): 6.9±1.2 vs. 5.1±0.7, 2.8±0.8, TNF-α (μg/L): 98.3±6.9 vs. 63.1±6.8, 30.2±2.1, all P <0.05]. Serum levels of NE, Fib and TNF-α in the death group were higher than those in the survival group [NE (μg/L):141.2±14.9 vs. 80.3±7.4, Fib (g/L): 7.6±1.5 vs. 5.7±1.0, TNF-α (μg/L): 105.4±7.8 vs. 68.2±4.6, all P < 0.05]. It was shown by ROC curve analysis that NE, Fib, TNF-α have some value in predicting the prognosis of children with severe pneumonia, the area under the ROC curve (AUC) were 0.889, 0.809, 0.803, 0.961, 95% confidence internal (95%CI) were 0.817-0.968、0.706-0.909、0.702-0.891、0.908-1.000, the sensitivity were 71.2%, 62.7%, 64.9%, 92.3%, the specificity were 73.5%, 68.3%, 74.5%, 90.9%, all P = 0.000. The PSI of severe pneumonia group was significantly higher than that of the general group (97.4±12.1 vs. 76.4±9.6), the PSI of the death group was obviously higher than that of the survival group (100.8±13.1 vs. 87.3±10.5), and the differences were statistically significant (both P < 0.01). Spearman correlation analyses showed that serum NE, Fib, TNF-α and PSI were significantly positively correlated in children with severe pneumonia respectively (r = 0.767, 0.734, 0.673, all P < 0.05), and there were positive correlations between NE and Fib (r = 0.655,P = 0.000), NE and TNF-α (r = 0.530,P = 0.000), Fib and TNF-α (r = 0.522,P = 0.000). Conclusion The combined detections of NE, Fib, and TNF-α levels can help clinicians determine the changes in the condition of children with severe pneumonia and evaluate their prognoses, combined detection has high sensitivity and specificity.

Objective: To explore the safety and effectiveness of 3D reconstruction in thoracoscopic posterior basal segmentectomy (S10). Methods: Between March 2018 to September 2018, 14 patients underwent thoracoscopic anatomical resection of the posterior basal segment of the lung (S10). Results: Of the 14 patients, including 5 males and 9 females, age (52.2±5.3)years, size (1.1±0.6)cm, 6 left S10 and 8 right S10. The number of pathological type of microinvasive adenocarcinoma, benign nodule, and metastatic carcinoma was 12, 1, and 1 cases. The average preoperative planning time was (44.9±5.7)min, and the average operation time was (134.8±26.3)min. The blood loss was (25.5±4.1)ml, with (8.1±2.7) lymphadenectomy, no positive metastasis. The coincidence rate of 3D reconstruction and intraoperative anastomosis in the tumor location, B10, A10, and V10 were 100%(14/14), 100%(14/14), 93%(13/14) and 71%(10/14). The median duration of chest tube insertion was (2.3±2.1)day. The incidence of postoperative complications was 21%(3/14), including 7%(1/14) of air leakage, 7%(1/14) of arrhythmia, 14%(2/14) of pulmonary infection, and 14%(2/14) of operation. All the cutting edge was >2 cm. There was no perioperative death, no conversion to thoracotomy or lobectomy. The mean follow-up time was (8.1±2.2)months. There were no recurrence, metastasis or death in the 14 patients. One patient had chronic cough and no hemoptysis. Conclusions Preoperative 3D reconstruction make the anatomic thoracoscopic posterior basal segmentectomy (S10) safer and more effective.