Objective To prepare a national reference standard for the quantification of HEK293 cell DNA content,so as to provide a support for the determination of residual DNA in HEK293 cells in the industry.Methods HEK293 cell DNA prepared using Genomic-tip 500/G and genomic DNA purification reagents was used as source materials,and the purity and content were assessed using ultraviolet spectrophotometry and agarose gel electrophoresis.After dilution to approximately 100 ng/μL,the DNA was aliquoted at 160 μL/tube.Five different laboratories were organized for collaborative calibration by using ultraviolet spectrophotometry, and the stability and applicability were evaluated.Results The HEK293 cell DNA national reference standard exhibited A_(260)/A_(280) ratios between 1.8 and 2.0 and displayed a single band on electrophoresis,meeting the specified criteria.Collaborative calibration across five laboratories yielded 78 valid data points with an average content of 104.8 ng/μL,a relative standard deviation(RSD) of 4.2%.The 95% confidence interval for the mean was 103.8—105.8 ng/μL,and the 95% reference range for single measurements was 96.0—113.6 ng/μL.The average confidence limit rate was 1.0%,and the recommended storage condition was-80 ℃.Applicability studies were conducted using two different models of fluorescence quantitative PCR instruments.The reference standard exhibited good applicability within the range of 0.3—3 000 pg/reaction,with amplification efficiencies of 101% and 95%,and R~2 values of 0.999 2 and 0.999 5 for the standard curves,respectively.Conclusion This batch of HEK293 cell DNA national reference standard meets all required specifications and can be utilized as a national reference standard for fluorescence quantitative PCR detection,with a certified content of 104.8 ng/μL,assigned batch number 270039-202301.

Objective To prepare a national reference standard for the quantification of HEK293 cell DNA content,so as to provide a support for the determination of residual DNA in HEK293 cells in the industry.Methods HEK293 cell DNA prepared using Genomic-tip 500/G and genomic DNA purification reagents was used as source materials,and the purity and content were assessed using ultraviolet spectrophotometry and agarose gel electrophoresis.After dilution to approximately 100 ng/μL,the DNA was aliquoted at 160 μL/tube.Five different laboratories were organized for collaborative calibration by using ultraviolet spectrophotometry, and the stability and applicability were evaluated.Results The HEK293 cell DNA national reference standard exhibited A_(260)/A_(280) ratios between 1.8 and 2.0 and displayed a single band on electrophoresis,meeting the specified criteria.Collaborative calibration across five laboratories yielded 78 valid data points with an average content of 104.8 ng/μL,a relative standard deviation(RSD) of 4.2%.The 95% confidence interval for the mean was 103.8—105.8 ng/μL,and the 95% reference range for single measurements was 96.0—113.6 ng/μL.The average confidence limit rate was 1.0%,and the recommended storage condition was-80 ℃.Applicability studies were conducted using two different models of fluorescence quantitative PCR instruments.The reference standard exhibited good applicability within the range of 0.3—3 000 pg/reaction,with amplification efficiencies of 101% and 95%,and R~2 values of 0.999 2 and 0.999 5 for the standard curves,respectively.Conclusion This batch of HEK293 cell DNA national reference standard meets all required specifications and can be utilized as a national reference standard for fluorescence quantitative PCR detection,with a certified content of 104.8 ng/μL,assigned batch number 270039-202301.

Objective To investigate the feasibility of using quantitative PCR(qPCR)technology to detect large fragments of host cell residual DNA(HCD)in recombinant adeno-associated virus(rAAV)gene therapy products.Methods Four different serotypes of rAAV were extracted for the nucleic acids,two fragment sequences of 244 bp and 562 bp within the long terminal repeat sequence(LTR)in the genome of host cells HEK293 were specifically quantified by qPCR,and the proportion of HCD in the total nucleic acids was calculated.Results Large fragments of HCD in qPCR quantifiable range were detected in four different serotype rAAV products,with the abundance ranging from 0. 3% to 5. 4%. As the length of the detected fragment increased,the abundance of HCD fragments showed a decreasing trend.Conclusion qPCR technology can be used to determine the presence of large fragments of HCD in rAAV products.

Objective To compare the application of two pretreatment methods,deoxyribonuclease(DNase)treatment and ligand affinity,in detecting the residual amount of free and mispackaged host cell DNA(HCDNA)in recombinant adenoassociated virus(rAAV).Methods Free and mispackaged HCDNA were isolated by DNase treatment and ligand affinity respectively,and then the nucleic acid was extracted and detected by qPCR. The accuracy and reproducibility of two pretreatment methods for detecting HCDNA residues were compared.Results Using DNase treatment,the result of nucleic acid quantitative detection in non-DNase-treated group was the total residual amount of HCDNA,that in DNase-treated group was the amount of mispackaged HCDNA,and the difference between them was the residual amount of free HCDNA. The recovery rates of both the untreated and treated groups were more than 75%,and the RSD of reproducibility was less than 30%. Using affinity extraction method,with the affinity ligand combined with rAAV,the result of recovery rate of mispackaged HCDNA was over 75%,and that of free HCDNA was only 36%.Conclusion DNase treatment method can effectively detect free and mispackaged HCDNA,laying a foundation for further research.

OBJECTIVE: To probe into status quo and tendency of medication in patients with leprosy. METHODS: 2 745 prescriptions collected from dispensary for western medicine during Aug. 2007～Jun. 2008 were analyzed statistically in respects of antibiotics and its combination, anti-bacterial drugs, antipyretic analgesic and anti-inflammatory drugs for top 3 diseases. RESULTS: The average cost of prescriptions was 12.88 yuan. Top 3 diseases in list of clinical diagnosis were as follows: leprosy ulcers, leprosy reactions and upper respiratory tract infection. 1 112 cases were treated with antibacterial drugs accounting for 44.13% in total cases; drug combination was applied in 150 cases accounting for 13.49% in antibiotics cases. CONCLUSION: Patients with leprosy are mainly treated with antipyretic analgesic, anti-inflammatory drugs, anti-bacterial drugs and glucocorticoid. Some inappropriate phenomena such as abuse of drug and irrational medication demand quality control of prescription.

Object　To determine the timing of NO expression in brain tissue after severely diffused brain injury．It would contribute to the understanding of the pathological and physiological functions of NO．Methods　On the basis of the Marmarou diffused brain injury model，the rat was killed at different hours afterward．and the NO expression in brain was measured by Griess reaction．Results　It was shown that NO in brain tissue increased quickly after trauma （5.20umol／100g），then decreased in 12h（1.96umol／100g）after　trauma，a secondary NO elevation appeared in 24h（2.31umol／100g） and 48h（2.69umol／100g） after DBI．Conclusion　It indicated that NO expression in brain tissue increased after trauma．As MCAO ischemia model，there was also NO increase at the intermediate and late stage，which might take part in the secondary pathological changes at late stage of cerebral injury．

Objective To evaluate the application value of the VATS combined with artificial pneumothorax in extended thymectomy. Methods From March 2013 to November 2014, we completed 45 cases of expanded thymectomy in patients with myasthenia gravis .According to the choice of patients , the surgeries were divided into two groups .The artificial pneumothorax group (24 cases) was given thoracoscopic expanded resection under artificial pneumothorax , while the conventional surgery group (21 cases) was given conventional thoracoscopic surgery .The operation time , intraoperative bleeding , operative field show ( to expose the offside mediastinal fat and cardiophrenic angle fat fully ) and symptom relief were compared between the two groups . Results The operations were successful in all the 45 cases.As compared with the conventional surgery group , the artificial pneumothorax group had shorter operation time [(93.8 ±16.8) min vs.(119.5 ±23.3) min, t=-4.293, P=0.000], less intraoperative hemorrhage [(54.2 ±43.7) ml vs.(92.9 ±41.0) ml, t=-3.048, P=0.004] and better operation exposure [91.7% (22/24) vs.57.1%(12/21),χ2 =7.228, P=0.007].However, there was no significant difference in symptom remission rate between the two groups . Conclusion VATS under artificial pneumothorax for thymus expanded resection can fully expose the operation field , with shorter operation time and less blood loss .

Neoadjuvant chemotherapy or neoadjuvant chemotherapy in combination with targeted therapy has been widely accepted as the standard treatment for locally advanced breast cancer (Ⅱb-Ⅲ). Nearly forty percent of the patients who accepted neoadjuvant chemotherapy achieved pathological complete response of axillary lymph nodes in addition to downstage the primary lesions. However, for patients with clinical complete response of lymph nodes after pre-operative systemic therapy, there are constant controversies regarding the prediction of axillary lymph nodes response and sentinel lymph nodes biopsy after the treatment. Here we design to review the latest studies about how to evaluate the axillary lymph nodes response after neoadjuvant chemotherapy and try to enlighten the treatment choices in clinical practice.

The overexpression of human epidermal growth factor receptor 2 (HER-2) is generally considered as an signiifcant predictor of poor prognosis, but the outcome has been rewritten with the appearance and application of the HER-2 targeted monoclonal antibody trastuzumab and chemotherapy plus targeted therapy. For the superiority of acting as in vivo susceptibility” test, neoadjuvant chemotherapy has become a new comprehensive treatment mode for operable breast cancer. And it has also provided an important approach to investigate the effectiveness of newly appeared targeted therapy. We focused more on reviewing and analyzing the results of clinical trials related to preoperation chemotherapy and the latest studies in HER-2 positive breast cancer in this article.

Objective To disclose the variant rule of HIV-1 vertical transmission by studying the differences of genotypes and quasispecies in the individuals with HIV-1 vertical transmission. Methods RNA was extracted from the plasma of the individuals of vertical transmission and C2-V5 DNA segment of HIV-1 env gene was acquired by RT-PCR. Purified DNA segment was inserted into T vector and transformed into Top10 Escherichia coli. Positive clones were acquired by blue-white screening and used as models in PCR. PCR products were analyzed by conformation sensitive gel electrophoresis (CSGE). The clones of major and minor quasispecies were sequenced. Results HIV-1 quasispecies in the mother of the first group were less complex than her child, and their quasispecies nucleotide sequences were highly homologous and of HIV-1 of C subtype. HIV-1 quasispecies in the mother of the second group were more complex than her child, and their quasispecies nucleotide sequences were highly homologous and of HIV-1 of AE subtype. Conclusion HIV-1 genetic subtypes are generally not changed in vertical transmission, but HIV-1 quasispecies could be selected in vertical transmission and screened by the new environment of the new host which may both induce the changes of dominative and minor quasispecies. We also find that complexity of HIV-1 quasispecies is associated with immune status of host.