Insect antifreeze protein (AFP) has high antifreeze activity. Antifreeze proteins can be used in cryopreservation of biological tissues and cells. We expressed an antifreeze protein from the desert beetle Microdera punctipennis in yeast and determined the function of the protein at low temperatures. Yeast expression vector, pPIC9K-Mpafp698, was constructed and transformed into Pichia pastoris GS115. The expression of MpAFP698 was induced by methanol, and identified by tricine SDS-PAGE and Western blotting. Mpafp698 gene was inserted into the genome of the host yeast strain GS115, and correctly expressed. Hardly any yeast's own protein was secreted into the media. Cryoprotective experiments showed that MpAFP698 can significantly protect mouse liver as well as other mouse organs from cold damage compared with those in the control of Bovine serum albumin (BSA) addition. Besides, the hemolysis of blood cells protected by MpAFP698 at 4 degrees C was reduced and the survival rate of SF9 cells protected by MpAFP698 after freezing and thawing was increased compared to those of the control with BSA addition. Our results showed that MpAFP698 can be expressed in yeast, which allows a convenient purification of the MpAFP protein that has the cryoprotective effect.
Animals ; Antifreeze Proteins ; biosynthesis ; Blotting, Western ; Cold Temperature ; Coleoptera ; Cryoprotective Agents ; chemistry ; Electrophoresis, Polyacrylamide Gel ; Freezing ; Insect Proteins ; biosynthesis ; Mice ; Pichia ; metabolism ; Sf9 Cells

Objective To investigate the clinical value of lasting methylene blue staining in precise hepatectomy.Methods The clinical data of 21 patients with liver cancer who received precise hepatectomy after methylene blue staining at General Hospital of PLA from February to August in 2009 were retrospectively analyzed.After the hepatic pedicle Was dissected,methylene blue WaS injected into the portal vein,and then the hepatic pedicle was ligated.Parenchymal division is initiated along the line of devascularization demarcated on Glisson capsule.Results The success rate of methylene blue staining Was 100%.Methylene blue retained in the parenchyma for(80±23)minutes.Right hepatectomy was performed on 2 patients,left hepatectomy on 1,right posterior lobectomy on 2,right anterior lobectomy on 3,left lateral lobectomy on 1,segmentectomy of segment Ⅷon 2,segmentectomy of segment Ⅶ on 3,segmentectomy of segment Ⅵ on 1,segmentectomy of segment Ⅳ on 2 and combined segmentectomy on 4.The mean volume of blood loss,incidence of postoperative complications and postoperative hospital stay were(236±6)ml,14%(3/21)and(12±3)days.Conclusions Ligation of hepatic pedicle after methylene blue injection has the advantages of high success rate and lasting staining of parenchyma of liver.Especially,this staining method contributes to improve the precision of hepatectomy by guiding the segment selection during parenchyma transection.

Objective To evaluate three-dimentional (3D) reconstruction technique and methylene blue staining in precise anatomic hepatectomy.Methods The clinical data of 12 patients with hepatocellular carcinoma who were admitted to the Chinese PLA General Hospital from February 2009 to August 2011 were retrospectively analyzed.The 3D reconstruction of the liver tumor and intrahepatic vessels were done based on the computed tomography data and magnetic resonance imaging data.The portal vein supplying the tumor and its anatomic relationship with adjacent vessels were evaluated.Precise anatomic hepatectomy was performed guided by sustained methylene blue staining.Results The accurate rate of 3D model of the portal triad was 12/12.The shape of target segments observed after methylene blue staining was consistent with the results of 3D evaluation.Two patients received hemihepatectomy,3 received lobectomy,5 received monosegmentectomy or subsegmentectomy,2 received multisegmentectomy.The mean tumor diameter,operation time,blood loss,postoperative hospital stay and complication rate were 5.6cm (2.5-16.0 cm),(150±24)minutes,(236±25)ml,(10±3)days and 2/12,respectively.After a median follow-up of 14 months,tumor recurrence was found in 2 patients,and 1 of them died of tumor progression.Conclusions The 3D reconstruction may contribute to precise evaluation of the anatomic relationship between the tumor and its adjacent vessels.The 3D technique combined with sustained methylene blue staining may significantly improve the accuracy of anatomic hepatectomy.

OBJECTIVETo study target killing of 5-FU drug-fast cancer cells with thymidylate synthase (TS) and p16 gene promoters inducting TK gene expression.

METHODSTS promoter was inserted to 5' end and p16 promoter inserted to 3' end of TK cDNA sequence, constructing recombinant plasmid of pXJ41. Human rectal cancer cell lines of HR-8348 and normal peripheral blood mononuclear cells (PBMC) were transfected with the recombinant plasmid. Plating efficiency was counted and survival rates of cells were tested with MTT method. And suppression rates of xenograft tumors in nude mice were examined.

RESULTSRecombinant pXJ41 with double promotors and TK gene was transfected into HR-8348, and positive expression of TS and TK was observed. The expression of TK gene was consistent with TS expression. Plating efficiency was 9/300, 92/300 in transfected HR-8348 and contrast cells respectively (t = 33.885, P < 0.01). Cancer cell growth rate reduced markedly in the transfected group. The suppression rate of xenograft tumor growth was 74.5%. With the recombinant pXJ41 to transfect PBMC, p16 expression was positive, but TK and TS expressions were negative. No damnification was observed in PBMC.

CONCLUSIONSTS and p16 double promoters are capable of inducting TK target killing of 5-FU drug-fast cancer cells, thus protecting normal cells and improving safety of gene therapy.

Animals ; Antimetabolites, Antineoplastic ; metabolism ; pharmacology ; therapeutic use ; Disease Models, Animal ; Drug Delivery Systems ; Fluorouracil ; metabolism ; pharmacology ; therapeutic use ; Gene Transfer Techniques ; Genes, p16 ; Genetic Therapy ; Humans ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Neoplasms, Experimental ; drug therapy ; Promoter Regions, Genetic ; Thymidine Kinase ; genetics ; Thymidylate Synthase ; genetics ; metabolism ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays

Objective Infected pancreatic necrosis is a serious complication of necrotizing pancreatitis. A method of minimally-invasive retroperitoneal infected pancreatic necrosectomy using percutaneous nephroscope was evaluated. Methods 21 patients with acute pancreatitis were treated in our hospital from June 2008 to August 2009. Among 13 patients who developed infected pancreatic necrosis, 6 underwent percutaneous catheter drainage by CT guidance. Then retroperitoneal infected pancreatic necrostectomy using percutaneous nephroscope along the sinus tract were performed after drainage for 5-36 d. Results In these 6 patients, 3 received percutaneous nephroscopic treatment one time, 2 two times and 1 three times. There were no operative mortality and morbidity except that 1 patient developed pseudocyst 6 months after operation. Conclusion Retroperitoneal pancreatic necrosectomy by percutaneous nephroscope is a safe, feasible, minimally-invasive and efficient method for treating infected pancreatic necrosis when the indication and occasion are suitable. This method would be a valid therapeutic option for treating necrotizing pancreatitis. However, further evaluation is necessary.

Objective There is little research on the relationship of gene polymorphism of CYP2C19 and clopidogrel response after percutaneous transluminal angioplasty and stenting ( PTAS) in patients with ischemic cerebrovascular disease.The study aimed to investigate the relationship between gene polymorphism and high on-treatment platelet reactivity ( HTPR ) after PTAS and 6 months of regular dual antiplatelet administration in patients. Methods A total of 145 Chinese patients treated with PTAS in our de-partment from January 2011 to March 2014 were enrolled in this study.According to the gene sequencing, patients were divided into wild-type group(CYP2C19*1/*1,69 cases) and mutation group(heterozygous mutation CYP2C19*1/*2 and homozygous mutation CYP2C19*2/*2, 76 cases).Patients received a 100mg/d aspirin and 75mg/d clopidogrel maintenance dose (MD) as dual anti-platelet therapy after PTAS.The clopidogrel inhibition effect was measured by thrombelastography ( TEG) system 6 months after PTAS. Routine cerebral artery digital subtraction angiography was applied to evaluate whether there was restenosis in stent and logistic regres-sion analysis was used to analyze the influential factors of HTPR after PTAS and clopidogrel adminstration. Results After 6 months'regular administration of clopidogrel after PTAS, the platelet adenosine diphosphate ( ADP ) receptor inhibition rates in wild-type group, heterozygous mutation and homozygous mutation group were respectively (58.43 ±21.98)%, (47.80 ±22.93)%, (37.53 ± 21.84)%.The platelet ADP receptor inhibition rate was significantly decreased compared with wild-type group(P=0.001).Carriers of at least one CYP2C19 loss-of-function ( LOF) allele had a higher frequency of clopidogrel HTPR (35.5% vs17.4 % for patients with and without LOF alleles, respectively;P=0.014) .Using multivariate logistic regression analysis, the carriage of CYP2C19 LOF alleles was an independent predictor of the post-procedure HTPR (OR=2.356, 95% CI:1.053-5.272, P=0.037).The rate of ISR was remarkably higher in patients with at least one CYP2C19*2 alleles compared with wild-type patients(11.8%vs 1.4%, P=0.019) . Conclusion In patients with ischemic cerebrovascular disease, the CYP2C19 LOF allele had significant impact on post-procedure clopidogrel HTPR and the prognosis of ISR after PTAS.

OBJECTIVETo study the effects of the traditional Chinese drugs, Shuganyishen, on Ishikawa cell line.

METHODRat blood serum was prepared by using Chinese drugs serum pharmacology method, in which, Shuganyishen was contained or not contained. Ishikawa cells were subjected into five groups: (1) normal cells group (NCG), (2) rat blood serum without Shuganyishen group (NSGG) as negative control, (3) Shuganyishen group (SGG), (4) Cisplatin group (DDPG) as positive control, and (5) Shuganyishen with Cisplatin group (S&CG). MTT assay was used to evaluate the inhibition effect on cell growth in each group. The mRNA expression of estrogen receptor alpha and beta (ERalpha and ERbeta) in each group was detected by RT-PCR.

RESULTEffective inhibition of cell growth was found in the groups of SGG and S&CG, respectively (P < 0.05), which had no significant difference from positive control by MTT. The mRNA expression of ERbeta was slightly going up, while the expression of ERalpha was hardly effected in SGG by RT-PCR. Interestingly in S&CG, the mRNA expression of ERalpha was significantly down-regulated (P < 0.05), but the mRNA expression of ERbeta remained no change. As the positive control, the mRNA expression of ERalpha and ERbeta was significantly down-regulated in DDPG (P < 0.5).

CONCLUSIONTraditional Chinese drugs, Shuganyishen, may inhibit Ishikawa cell growth and had no effects on the expression of ERalpha, furthermore, can reverse the inhibition of ERbeta expression by Cisplatin.

Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Endometrial Neoplasms ; drug therapy ; genetics ; metabolism ; physiopathology ; Female ; Gene Expression ; drug effects ; ethics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Estrogen ; genetics ; metabolism

Objective To rapidly evaluate the efficacy,safety,and economics of dexmedetomidine in patients on mechanical ventilation in intensive care unit(ICU),and to provide references for clinical and decision makers.Methods To search PubMed,Embase,Cochrane Library,CNKI,WanFang Data databases and official websites of domestic and foreign Health Technology assessment(HTA)institutions from the inception to June 14,2023.HTA reports,systematic reviews/Meta-analyses,and pharmacoeconomic studies of patients with mechanical ventilation in ICU compared with dexmedetomidine versus placebo or other traditional sedatives were included.Two reviewers independently identified studies,extracted data,assessed the quality of included studies,and descriptive analyzed and summarised the results.Results A total of 13 literatures were included,including 10 systematic reviews/Meta-analyses and 3 economic studies.The analysis results of effectiveness showed that compared with placebo or other traditional sedatives,the use of dexmedetomidine in the sedation of mechanically ventilated patients in the ICU was associated with a shorter duration of mechanical ventilation and a shorter length of stay in the ICU.The analysis results of safety displayed treatment with dexmedetomidine could reduce the incidence of delirium,but may increase the incidence of bradycardia.The results of the economic analysis showed that the dexmedetomidine group had a higher cost of medication,but a lower overall ICU cost.Conclusion Dexmedetomidine has good efficacy,safety and economics in the sedation of patients on mechanical ventilation in ICU,but the economic studies included in the search were mainly foreign studies,and domestic economy studies needs to be further demonstrated.

Objective:To investigate key differential expressed genes(DEGs),enriched biological functions and signaling pathways in exosomes derived from angiosarcoma cells ISOHAS and angiosarcoma stem-like cells Sphere by bioinformatics analysis,to provide new therapeutic targets for angiosarcoma.Methods:Transcriptome sequencing of exosomes derived from ISOHAS and Sphere were performed to screen for DEGs by|log2FC|>2 and FDR<0.05 as criteria.Bioinformatics analysis was used to enrich GO and KEGG pathways of DEGs to identify biological functions and signaling pathways of enriched DEGs.STRING database was used to screen key DEGs,and iPath was used to visualize to identify metabolic pathways enriched by DEGs.Results:Transcriptome sequencing results showed that 91 DEGs were identified in exosomes derived from ISOHAS and Sphere.GO and KEGG pathway enrichment analysis demonstrated that main biological functions and signaling pathways enriched by DEGs were response to glucocorticoid and TNF signaling pathway,respectively.STRING database demonstrated that TNF and IL-6 were key DEGs.iPath metabolic network analysis demonstrated that DEGs were mainly identified in lipid metabolism and nucleotide metabolism.Conclusion:Sphere-derived exosomes may influence occurrence and development of angiosarcoma by carrying key genes TNF and IL-6 to interfere with glucocorticoid response,TNF signaling pathway,lipid metabolism,nucleotide metabolism and other biological functions and signaling pathways,providing new ideas for therapeutic targets for angiosarcoma.

Objective: To investigate the current basic situation of the staff of ultrasound departments in Shanghai′s medical institutions, for providing references in making management policy of these professionals. Methods: Questionnaire surveys on human resource and service ability were made to all the ultrasound departments of medical institutions in Shanghai in December 2013 and November 2018 respectively. Data of the two surveys were compared and analyzed, and were descriptively analyzed by mean and percentage. Results: The number of ultrasound professionals per 10 000 people in Shanghai was 1.04 in 2018. Tertiary hospitals had advantages in the number of the professionals, and the proportion of professional qualification, age, education background and professional title of the professionals. Compared to those data in 2013, the number of ultrasound professionals had increased 31.8% in 2018. The proportion of medical practitioners with medical imaging specialty was 95.6%(2 063/2 158), and had increased by 4.7 percent. The medical services workload of ultrasound was 19.82 million person-time, and had increased 45.8%. Conclusions Development of ultrasound departments was rapid, but the development of professionals was unbalanced with the development of medical services. It is suggested to strengthen training of ultrasound professionals and improve the system of hierarchical medical system.