BACKGROUND:Studies have shown that human leukocyte antigen (HLA)-A*0206 subtype is related to the abscess of nasopharyngeal carcinoma, but there is no corresponding transgenic animal models that could used to judge the relationship between HLA-A*0206 and nasopharyngeal carcinoma on the overal level and further research of immunotherapy and gene therapy. OBJECTIVE:To construct lentiviral vectors carrying pLVX-CMV-HLA-A*0206-HA-mCMV-ZsGreen and establish HLA-A*0206 transgenic mice. METHODS:The HLA-A*0206 sequence was synthesized. EcoRI recognition site was introduced in the 5’ end by polymerase chain reaction, and influenza virus hemagglutinin labels and BamHI recognition site were introduced in the 3’ end. Eco RI and Bam HI double enzyme digestion target fragments and the pLVX-CMV-mCMV-ZsGreen plasmids were connected to the digested productions and transfected JM109 competent cel s. The positive clones were selected and identified by double enzyme digestion and sequencing. The positive plasmid and packaging plasmids were transfected into 293T cel s, which were human renal epithelial cel line that can express SV40 large T antigen. The lentivirus containing target sequence was produced. RESULTS AND CONCLUSION:Gel electrophoresis and sequencing results showed that, HLA-A*0206 was successful y inserted into pLVX-CMV-mCMV-ZsGreen frame plasmids. Transfection efficiency was 92%after 48 hours of transfecting 293T cel s. The viral suspension titer was 5 × 108 measured by fluorescence method. Experimental findings indicate that, the lentivirus containing cytomegalovirus promoter, HLA-A*0206, influenza virus hemagglutinin label and Zsgreen report gene was successful y constructed.

Parkinson′s disease(PD)is a common disease caused by multiple factors and characterized by pathological degen?eration in the dopaminergic neural system. Based on its pathogenic factors,PD can be divided into several subtypes,so it is essential to develop therapeutic agents based on the main pathogenic factor of each subtype of PD. Recently it is confirmed that the mutation of leucine-rich repeat kinase 2(LRRK2)gene leads to increased activity of the LRRK2 notably,and then causes neurodegeneration. Thus developing LRRK2 inhibitors to modulate the kinase activity will be a novel therapy for the PD subtype which is caused by LRRK2 gene mutation. LRRK2,either a kinase or a GTPase,has two drug binding sites. Therefore,two types of LRRK2 inhibitors are being studied,one is the kinase inhibitor and the other is GTPase inhibitor. This paper summarizes the recent progress in the dis?covery and development of LRRK2 inhibitors.

Objective To accurately determine the biological characteristics , especially the disease-related indexes of SPF rabbits , and their gender differences .Methods A total of thirty 70-80 day old SPF New Zealand rabbits (male :female=1:1) were fed for a week, and then we weighed the body weight and main organs , determined the blood physiological and biochemical indexes , blood gas and arterial pressure , and measured the ventricular pressure by thoracotomy under respiratory support .Results Comparison of the male and female SPF New Zealand rabbits showed that there were significant differences in the mass of thyroid , adrenal gland, and liver (P<0.05 or P<0.01); the brain, pituitary gland, thyroid gland organ coefficients ( P<0.05 or P<0.01); the thyroid/brain, adrenal/brain, and liver/brain ratios (P<0.05 or P<0.01);the mean erythrocyte volume and mean hemoglobin content (P<0.05 or P<0.01);and in glutamyl transpetidase and amylase ( P<0.05 or P<0.01 ) , but no significant differences in blood gas analysis ,heart rate and carotid artery systolic and diastolic blood pressure , left ventricular systolic and diastolic blood pressure , and right ventricular systolic and diastolic blood pressure .Conclusions The gender has an impact on organ weight and blood physiological and biochemical indexes in New Zealand rabbits , but no significant influence on blood gas , blood pressure , heart rate and ventricular pressure in the rabbits .

Objective:According to the tumor of children psychology, explore a set of suitable for children accurate tumor radiotherapy positioning method, in order to improve the positioning accuracy, thus improve the tumor clinical cure rate.Methods: 1.Eliminate fear; 2.Adaptability training; 3.The methods of psychological suggestion. Evaluation method: take psychological induction of radiation induced by radiation therapy in children with and without taking mental children from the placement time, downtime in drug utilization rate, treatment rate and bit error rate comparison.Results: There is obvious difference between two groups (P<0.05).Conclusion: radiotherapy positioning of children with psychological intervention in the method, can improve the treatment of children's compliance.

Objective To explore the significance of core-needle biopsy ( CNB) in the diagnosis of thyroid lymphoma. Methods A case of Hashimoto's thyroiditis for several decades, showed a thickening of the neck for several months, and no abnormalities were found by the method of fine-needle aspiration( FNA), then we performed CNB and flow cytology. Results Thyroid lymphoma was finally diagnosed through CNB and flow cytology. Conclusion Thyroid lymphoma should be considered when the neck became thickened in short time in patients with chronic Hashimoto's thyroiditis. Core-needle biopsy accompanied with flow cytometry and immunohistochemistry analysis should be suggested as routine diagnostic method especilly when fine-needle aspiration was negative.

Objective To evaluate the inhibitory activity of Herba Taraxaci extract on Escherichia coli DH5α (E. coli DH5α) and to investigate proteomic response of E. coli. Methods Medicinal powder of Herba Taraxaci was extracted with the solvents of different polarity ( n-hexane, ethyl acetate, distilled water) , and then the obtained 8 different extracts were subjected to thin layer chromatography ( TLC) analysis. Microdilution method was performed to detect the minimum inhibitory concentration ( MIC) of different extracts and the growth curves were described. The protein expression profiles of E . coli treated with the extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis ( SDS-PAGE) and two dimensional electrophoresis (2-DE) . Results Water decoction of Herba Taraxaci could obviously suppress the growth of E. coli with a MIC of 1.95 mg/mL. The different extractions exhibited no antibacterial activity except ethyl acetate phase 3 with a MIC of 0.13 mg/mL, which was equal to 19.23 mg/mL of crude drugs. The results of TLC analysis showed that chlorogenic acid was undetectable in n-hexane extract and ethyl acetate phase 1 extract, and ethyl acetate phase 2 and 3 extracts showed obviously increased spots. The results of SDS-PAGE and 2-DE showed that water decoction of Herba Taraxaci had inhibitory effect on the expression of functional protein. The results of 2-DE showed that after treatment with ethyl acetate phase 3 at the concentration of 2 × MIC for 21 hours, the amount of protein spots were 92 less than those of the blank control group, the spots of E. coli DH5α soluble protein with expression amount down-regulated doubly were 24, and those with expression amount up-regulated doubly were 19. Ethyl acetate phase 3 extract had an effect on down-regulating the protein expression of E. coli DH5α soluble protein pH3-10, and water decoction of Herba Taraxaci had inhibitory effect on E. coli DH5αprotein expression. Conclusion Herba Taraxaci has significant antibacterial activity on E. coli DH5α, and the water-soluble fraction of chlorogenic acid and caffeic acid might be the active components. The possible antibacterial mechanism may be related with the regulation of bacterial protein expression.

[ ABSTRACT] AIM: To explore the relationship between FRAS 1 protein and brain metastases of non-small cell lung cancer (NSCLC).METHODS:The mRNA expression of FRAS1 in the brain metastatic tumor tissues and primary tumor tissues of NSCLC was detected by qPCR .The protein expression of FRAS 1 in the tumor tissues and normal tissues adjacent to tumor tissues of NSCLC was measured by SP method of immunohistochemistry .The protein expression of FRAS 1 in NSCLC primary tumor tissues with or without brain metastases was also determined .RESULTS:The mRNA expression of FRAS1 in the brain metastatic zone was nearly 10 times higher than that in the primary tumor tissues , and there was sig-nificant difference between the 2 groups (P<0.05).FRAS1 protein was expressed in the NSCLC primary tumor tissues , but was not found in the normal tissues adjacent to primary tumor tissues .The protein expression of FRAS 1 in the NSCLC with brain metastases was significantly higher than that without brain metastases ( P<0.01 ) .CONCLUSION: FRAS1 protein may be associated with the occurrence of NSCLC .The over-expression of FRAS1 protein may be related to brain metastases with NSCLC .

We obtained recombinant ferritin from Dermatophagoides f arinae ,and analyzed the characterization of the pro‐tein .A pair of primers was designed according to the known sequence of ferritin gene .The live mites identified and cultured lo‐cally were picked and the total RNA was extracted .The ferritin gene fragment was amplified by RT‐PCR ,and cloned into pET32a vector ,and then transferred into E .coli Top10 .The target gene obtained from the recombinant plasmid by digestion with Bam HⅠand Hind Ⅲ was connected to the prokaryotic expression vector pET‐32a .The expressed recombinant plasmid containing ferritin gene was constructed by cloning target gene into pET‐32a and transferred into E .coli Bl21 (DE3) .The ex‐pressed recombinant protein was analyzed by SDS‐PAGE ,and was purified by immobilized metal ion affinity chromatography (IMAC) .The ferritin expressed by dust mite was analyzed by the method of bioinformatics .The recombinant plasmid pET32a‐ferritin was constructed .SDS‐PAGE showed a correct molecular weight of the recombinant ferritin protein .After purification by affinity chromatography ,the protein showed only one strip on SDS‐PAGE gel .SDS‐PAGE showed a band at 20 kD .Dust mite ferritin contains 8 serine kinase ,7 threonine kinase ,7 tyrosine kinase ,and 0 histidine kinase phosphorylation sites .Hy‐drophilic region is larger than the hydrophobic region and it is an unstable protein .In conclusion ,the ferritin gene has been cloned and expressed .The purified ferritin has high purity . The study provides a basis for further study of composition and physicochemical properties of house dust mite allergen .

A method for the determination of cyromazine and dicyclanil residues on greasy wool was developed with HPLC and confirmed with HPLC-MS/MS.The cyromazine and dicyclanil residues on greasy wool were extracted with 1% trichloroacetic acid solution with ultrasonic, and cleaned up by MCX SPE column.The HPLC separation was performed on a Hypersil NH_2 using water-acetonitrile (V/V) as the mobile phase with gradient elution and detected at the wavelength of 214 nm.The corroboration method of HPLC-MS/MS was used with electro-spray ionization of positive ion mode.The [ M + H ]~+ and characteristic ions of dicyclanil were m/z 191.0, 150.0 and 163.0, and cyromazine were m/z 167.0, 85.0 and 125.0.The linear ranges of cyromazine and dicyclanil were 0.05-5.0 mg/L.There were good linear relationships between the peak area and concentration in the linear range.The correlation coefficient was 0.9999.The detection limit of cyroma zine was 0.02 mg/kg, and dicyclanil was 0.01 mg/kg.The average recoveries of cyromazine and the dicycla nil were 95.0%-99.9% and 83.6%-92.2%, respectively.

The clustered regulatory interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system is the part of the prokaryotic immune system, which could recognize and delete the exogenous sequences originated from virus or plasmid. Based on its mechanism, CRISPR-Cas9 system was developed into the new generation of gene editing tool. Compared to the existed technologies such as ES targeting, ZFN or TALEN, CRISPR-Cas9 system is a more efficient, economical and promising approach to manipulate the genome. In this review, we summarize the research progress about CRISPR-Cas9 technology, especially the latest applications in gene therapy studies of human diseases.
CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genetic Therapy ; Humans ; Plasmids