OBJECTIVESTo investigate whether antisense human telomerase reverse transcriptase (hTERT) could inhibit the activity of telomerase and the proliferation of K562 cells.

METHODSThe antisense plasmid was constructed by reverse insertion of hTERT PCR product into plasmid pLNCX-neo. Then the constructed plasmid was introduced into K562 cells by liposomes-mediated DNA transfection. The inhibition effects of telomerase on the proliferation of K562 cells were analyzed by MTT and colony formation assay, the telomerase activity of K562 cells by TRAP-PCR ELISA methods.

RESULTSThe growth rate of antisense hTERT transfected K562 cells was significantly lower than those of the controls, and the colony formation capacity of the transfected cells decreased significantly (P < 0.01), the colony number is (100.33 +/- 7.57)/10(3) cells, (92.67 +/- 5.86)/10(3) cells and (50.33 +/- 6.11)/10(3) cells for control K562 cells, K562 neo cells and antisense hTERT transfected HL60 cells, respectively. The telomerase activity of antisense hTERT transfected K562 cells was significantly inhibited.

CONCLUSIONThe expression of an antisense sequence to the mRNA sequence of telomerase protein subunit can inhibit the activity of telomerase, slow the cell growth and inhibit the capacity of colony formation of K562 cells.

Cell Division ; drug effects ; Humans ; K562 Cells ; Plasmids ; genetics ; RNA, Antisense ; genetics ; pharmacology ; RNA, Messenger ; genetics ; Telomerase ; drug effects ; genetics ; metabolism ; Transfection

Objective To explore the expressions of hypoxia inducible factor ( HIF), vascular endothelial growth factor receptor 2 ( VEGFR 2), and microvessel density ( MVD) in adrenocortical adenoma ( ACA) and adrenocortical carcinoma ( ACC), in order to discuss their potential role in the development of adrenal tumours. Methods Fifty-five adrenal tumour specimens resected in the hospital with complete clinical data (including 30 ACA cases and 25 ACC cases) were examined by immunohistochemistry for the expressions of HIF-2α, HIF-1α, VEGFR 2, and MVD. Results VEGFR 2 and MVD up-regulated were found in the ACC group (P＜0.05). The expression of HIF-2α and HIF-1α correlated with VEGFR 2 (P＜0.05). The expressions of VEGFR 2 and MVD were related to some clinicopathological features ( P＜0. 05 ). Additionally, tumour size, expression of VEGFR 2 and MVD were independently associated with ACC (P＜0.05). Conclusions The high expression of HIF-2α, VEGFR 2, and MVD in adrenal tumours suggested their roles in tumour angiogenesis, which indicated that anti-angiogenesis therapies deserve intensive studies for malignant adrenocortical tumours.

Objective To explore the molecular mechanism of pituitary growth hormone adenoma cell proliferation.Methods Functional growth hormone-secreting pituitary adenoma(fGH-PA)tissue samples were collected from 12 patients with acromegaly.The exchange protein 1 directly activated by cAMP(Epac1)mRNA expression levels in fGH-PA tissues and rat RGC-5,MMQ and GH3 cells were determined by qPCR.The expression levels of Epac1 in fGH-PA tissues were determined by immunohistochemistry.Western blot was used to determine the expression levels of Epac1 in MMQ and GH3 cells.Overexpression or knockdown of Epac1,or knockdown of cAMP response element-binding protein(CREB)in GH3 cells,cell cycle changes were determined by flow cytometry,cell proliferation ability was determined by CCK-8 assay,and the expression levels of p-CREB,CREB,Cyclin D1,CDK2 and p21 in cells were determined by Western blot.Results qPCR and immunohistochemistry results showed that the expression levels of Epac1 mRNA and protein in fGH-PA tissues were significantly higher than those in adjacent normal tissues(P<0.05).qPCR and Western blot results showed that compared with RGC-5 cells,the expression levels of Epac1 mRNA and protein in MMQ and GH3 cells were significantly increased(P<0.05).After overexpres-sion of Epac1 in GH3 cells,compared with the Control group,the proportion of cells in G0/G1 phase and S phase in the Epac1-OE group were significantly reduced(P<0.05),and the proportion of cells in G2/M phase were significantly increased(P<0.05);the cell prolifera-tion ability were significantly enhanced(P<0.05);the expression levels of p-CREB and Cyclin D1 in cells were significantly increased(P<0.05),the expression levels of CDK2 and p21 were significantly decreased(P<0.05),while there was no significant change in the expression level of CREB between the two groups(P>0.05).After knockdown of Epac1 or knockdown of CREB in GH3 cells,all of the above results were reversed(P<0.05).Conclusion The overexpression of Epac1 in pituitary growth hormone adenoma cells can up-regulate the levels of p-CREB in cells,and promote adenoma cells to pass through G1/S phase checkpoint,resulting in cell cycle checkpoint disorder and massive proliferation,but it does not affect the expression levels of CREB.

Objective To investigate the characteristics of MRSA colonization and their influence to the prognosis of patients in respiratory intensive care unit (RICU),so as to provide the basis for the caring of the RICU patients.Methods A prospective study included 246 cases of patients admitted to RICU in our hospital during Jan 2010 to Neb 2011.Swab specimens from nares,pharynx,axillae,groin,chest and hands of the patients were collected and cultured at the first day in RICU,and then repeated every 3 days.The patients were divided into MRSA colonization group (n =30) and MRSA non-colonization group (n =216) according to MRSA culture results.Results The MRSA colonization rate was 12.19％ in RICU patients,including 16 cases of hospital-acquired colonization.No significant difference was found in the gender,the APACHE Ⅱ score and APACHE Ⅲ between MRSA colonization group and the MRSA non-colonization group (P ＞ 0.05).The mean age in MRSA colonization group was significantly greater than that of the MRSA non-colonization group [(81.13 ± 6.86) vs (74.78 ± 11.89) ; t =2.955,P =0.003)] ; The number of hospitalization and the mortality in one year of the MRSA colonization group was significantly more than those of MRSA non-colonization group [(1.83 ±1.216) vs (1.06 ±0.257),40.0％ vs 16.7％ ;t =2.998,x2 =9.132;P＜0.01)].MRSA was mainly colonized in nares.Conclusions MRSA colonization has an important impact on the prognosis of RICU patients.More than 50％ of MRSA colonization cases are hospital-associated,suggesting that we should strengthen the bedside care,prevention of cross infection.

Background It has been reported that orthokeratology has the effects of slowing down myopia progression and axial elongation.However,the affecting mechanism of orthokeratology wearing on ocular peripheral refraction is still not elucidated.Objective This study was to observe and compare the changes of ocular peripheral refraction and relative peripheral refraction (RPR) in low to moderate myopic eyes of children after wearing orthokeratology lens and spectacles for 6 months.Methods A randomized controlled clinical trial was carried out after approval of Ethic Committee of Beijing Tongren Hospital and informed consent of guardians of the children.One hundred myopic children aged (ll.0±1.9) years were recruited in Beijing Tongren Hospital from June 2014 to January 2015,with the diopter of-0.50 to-6.00 D.The subjects were randomized into orthokeratology group and spectacles group by the process PLAN PROC of software SAS 9.1.3,50 for each group.The subjects in the orthokeratology group wore orthokeratology lens for 6 months and those in the spectacles group wore spectacles for the same period.An infrared open-field autorefractor was employed to measure the refraction at central 0°,temporal 15°,temporal 30°,nasal 15°and nasal 30° radial lines before and after wearing lens for the assessment and comparison of the changes of peripheral refraction and RPR.Results There was no significant difference in spherical equivalent between the orthokeratology group and the spectacles group before wearing lens ([-3.35±1.31] D versus [-3.01± 1.15] D,P =0.20).The peripheral refraction values in the orthokeratology group were (-2.28 ± 1.60),(-3.28±1.41),(-3.40±1.23),(-3.38±1.12) and (-2.09±1.29)D at nasal 15°and nasal30°,central,temporal 15° and temporal 30°radial lines before wearing lens,and reduced by (0.29±1.67),(0.85±1.66),(0.92±1.76) and (0.66±1.66) D at nasal 30°,nasal 15°,central and temporal 15° after wearing lens,respectively,with significant differences at nasal 15°,central and temporal 15°(all at P＜0.05).The peripheral refraction values in the spectacles group were (-1.88±1.30),(-2.66±1.18),(-2.89±1.27) and (-1.94±1.31)D at nasal 15°,nasal 30°,temporal 15 ° and temporal 30°,radial lines before wearing lens and increased by (-0.25±0.80),(-0.43 ±0.67),(-0.32±0.64) and (-0.22±0.75)D after wearing lens,respectively,with significant differences between before and after wearing lens (all at P＜0.05).The RPR shifted from hyperopia defocus to myopia defocus before and after wearing lens in temporal 15° and 30° radial lines in the orthokeratology group,and at various radial lines in the spectacles group,the RPR showed gradually worsening of hyperopia defocus.Conclusions Long-term wearing of orthokeratology results in a hyperopia shifting in myopic children by exposing the peripheral retina towards relative myopia defocus,whereas wearing spectacles leads to a relative hyperopia defocus on retina.Thus,orthokeratology may slow down the myopia progression.

Heterotypic cell-in-cell structures(heCICs)mediate unique non-autonomous cell death,which are widely involved in a variety of important pathological processes,such as tumorigenesis,pro-gression and clinical prognosis.Methylenetetrahydrofolata dehydrogenase 2(MTHFD2),one of the key enzymes of one-carbon metabolism,is highly expressed in a variety of tumor cells.In this study,in order to investigate the effect of MTHFD2 on the formation of heCICs,liver cancer cells and immune cells were first labeled separately by live cell dyes,and the heCIC model was established by using fluorescence mi-croscopy for cell imaging and analysis.After transiently knocking down MTHFD2 in cells by RNAi,we found that the ability of PLC/PRF/5 and Hep3B to form heCICs with immune cells was significantly in-creased(all P＜0.01).MTHFD2 recombinant expression plasmid was constructed by the homologous re-combination method,and MTHFD2 overexpression cell lines were further constructed.Then,the effect of MTHFD2 overexpression on the ability to form heCICs was detected by co-culturing the overexpression cell lines with immune cells.The results showed that the rate of heCIC formation was significantly re-duced after overexpression of MTHFD2(all P＜0.001).In conclusion,this study demonstrated that MTHFD2 is a negative regulator of heCIC formation,providing a research basis for targeting MTHFD2 to promote heCIC formation and enhance the in-cell killing of immune cells.

In this experiment, the PK/PD fitting model of Chuanxiong(Chuanxiong Rhizoma) in the treatment of rheumatoid arthritis was established in the form of acupoint combined with external application gel paste. Firstly, the rheumatoid arthritis model was induced by ovalbumin, and the articular fluid of rabbits was extracted by microdialysis. The pharmacokinetic process of Chuanxiong in rabbit articular fluid was analyzed by UPLC-MS/MS, and the pharmacokinetic model was established. The pharmacodynamic effects of Chuanxiong on inflammatory factors IL-1β, TNF-α, and IL-6 were analyzed by enzyme-linked immunosorbent assay(ELISA). The pharmacodynamic model was established, and the PK/PD model was obtained by fitting the data of pharmacokinetics and pharmacodynamics. The results of pharmacokinetics showed that the concentration of ligustrolide A in the articular cavity by drug administration on classical acupoint Zusanli(ST 36) was higher than that by Yanglingquan(GB 34), which reflected the advantage of typical acupoint, while ligustrazine concentration was higher after administration through Yanglingquan than through Zusanli, which was different from the traditional acupoint theory. The results of pharmacodynamics showed that the drug had lag effect. The PK/PD model was constructed by fitting the data. When IL-1β was taken as the efficacy index, the PK/PD models of Chuanxiong in typical acupoint Zusanli group, atypical acupoint Yanglingquan group, and non-acupoint group were E=115.28C_e/(3 316.72+C_e), E=108.73C_e/(2 993.47+C_e), and E=101.34C_e/(3 028.51+C_e). When TNF-α was taken as the efficacy index, the PK/PD models of Chuanxiong in typical acupoint Zusanli group, atypical acupoint Yanglingquan group, and non-acupoint group were E=68.31C_e/(3 285.16+C_e), E=59.27C_e/(2 919.86+C_e), and E=53.61C_e/(2 862.87+C_e). When IL-6 was taken as the efficacy index, the PK/PD models of Chuanxiong in typical acupoint Zusanli group, atypical acupoint Yanglingquan group, and non-acupoint group were E=59.92C_e/(3 461.17+C_e), E=58.34C_e/(2 723.51+C_e), and E=49.17C_e/(2 862.76+C_e). The parameters showed that there were significant differences in E_(max), EC_(e50) and k_(eo). The analysis of data found that the PK/PD fitting effect of Zusanli, a typical acupoint, was the best, which proved that it was still the best site for drug administration. To sum up, it shows that there may be bidirectional selectivity between drugs and acupoints.
Animals ; Rabbits ; Tumor Necrosis Factor-alpha ; Chromatography, Liquid ; Interleukin-6 ; Tandem Mass Spectrometry ; Acupuncture Points ; Arthritis, Rheumatoid/drug therapy*

Objective To use World Health Organization Family International Classifications (WHO-FICs) to explore the framework and approaches of development and research of guidelines of rehabilitation at levels of policies, community and services.Methods The important documents and tools of rehabilitation at international level, including United Nations Convention on the Rights of Persons with Disabilities, WHO World Report on Disability, Community-based Rehabilitation Guideline, Rehabilitation in Health Service System, and International Classification of Diseases (ICD), International Classification of Functioning, Disability and Health (ICF), and International Classification of Health Intervention (ICHI) of WHO-FICs, had been discussed.Results The framework, classifications, diagnosis and description of diseases and functioning, coding, intervention and functioning evaluation based on ICD-11, ICF and ICHI-β-2 had been established for development and implementation of rehabilitation guidelines and Cochrane rehabilitation.Conclusion The framework and systematic approaches of ontology, classification, terminology, coding, diagnosis and description of diseases and functioning, interventions and evaluations for the development and implementation of rehabilitation guidelines had been developed.