BACKGROUND: Drug treatment has unsatisfactory effect on Alzheimer disease, while many studies have indicated that the transplantation of bone marrow stromal cells (MSCs) is effective on Parkinson disease, cerebral ischemia, etc., but the mechanism is still unclear.OBJECTIVE: To imitate transplantation environment by co-culture of amyloid β1-40 (Aβ1-40) damaged PC12 cells and MSCs, observe the effect of bi-directional information feedback in the microenvironment on the transdifferentiation of MSCs to nerve cells, and observe its protective effect on the apoptosis of damaged PC12 cells.DESTGN: A comparative observation.SETTING: Department of Neurology, China Medical University.MATERIALS: SD rats of 2-3 weeks after birth either male or female were used. PC12 cell lines were purchased from the Institute of Cell Biology, Chinese Academy of Sciences; neuro-specific enolase (1:50, Boster, Wuhan);Methyl-thiazol-tetrazolium (MTT) 15 μL (terminal concentration of 0.5 g/L).METHODS: The experiment was carried out in the Experimental Center (provincial experimental animal center) of China Medical University from June to July in 2004. Bilateral femurs were aseptically removed from 1 SD rat, and MSCs were identified using CD44 antibody immunofiuorescently. PC12 was used to replace nerve cells. The PC12 cells were stimulated by Aβ1-40 then transferred by Transwell. There were 5 groups: Group A: normally cultured PC12 co-cultured with MSCs; Group B: Aβ1-40 stimulated PC12 co-cultured with MSCs; Group C: normal PC12 supernatant+MSCs; Group D: damaged PC12 supernatant+MSCs; Group E: MSCs cultured with common medium 1640.MAIN OUTCOME MEASURES:Routine immunohistochemical staining was performed. NSE positive cells were observed under inverted fluorescence microscope, 10 visual fields (200×) were randomly selected to count the positive cells. MTT metabolic rate was used to detect the proliferation of MSCs in each group. The differences of measurement data were compared using the one-way analysis of variance.fluorescent and bright fields, NSE positive cells appeared as red fluorescence, MSCs were bipolar, multipolar and cone shapes, and appeared as neuron-like forms with dendrite-like structure, and there were extensive connections among some neuron-like cells. The NSE positive rates was obviously higher in group B than groups A, C, D and E (P ＜ 0.01 ).in groups A, C, D and E (F=9.713, P＜ 0.01).

Objective To establish the co-culture system of marrow stromal cells (MSCs)and A?1-40 injured PC12 in vitro and to evaluate the effect and mechanisms of the system inhibiting apoptosis of PC12 induced by A?1-40.Methods MSCs and PC12 were cultured in vitro and identified by CD44 immunofluorescent staining;PC12 were damaged by A?1-40,and transferred by transwell followed by the classification into 5 groups. PI and Annexin-V co-fluorescent staining was performed,then PC12 apoptosis were detected by flow cytometry and EM;Supernatant was analyzed by enzyme-linked immunoadsordent assay (ELISA) to detected TGF-?,NGF,BDNF,and bFGF. Results About 96% MSCs showed CD44 positive cells. Co-culture group had the lowest rate of PC12 apoptosis(46.17%?8.28%,F=61.637,P0.05). Conclusion The co-culture system of MSCs and A?1-40 injured PC12 in vitro could inhibit apoptosis of PC12 induced by A?1-40. Thus grafted MSCs have the possibility to inhibit neuronal apoptosis by A?in the diseased brain.

Objective To compare magnetic beads kit,agrose gel recovery kit and heparinase I three methods to purify the micro DNA from crude heparin, then use q-PCR to identify the species origins and select the best method.Methods Using magnetic beads kit,agrose gel recovery kit and heparinase I to purify micro DNA from crude heparin and combined the porcine,bovine and ovine identification kits to identify the species origins and conformed the minimum detection limit of different percentage of ovine crude heparin in porcine crude heparin.Results Three pretreatment methods all can solve the pretreatment difficulties and we found that the haparinase was the best method; the minimum detection limit was 0.01%of ovine crude heparin in porcine crude heparin.Conclusion The heparinase method is the best pretreatment method and can successfully solve the pretreatment difficulties.Heparinase combine the porcine, bovine and ovine identification kits can identify the species origins from crude heparin.

Objective To develop and verify a method for determination of residual host cell DNA in recombinant human interferon α2b substances, which is used for the quality control of the product.Methods The residual host cell DNA was extracted by wako DNA extractor kit and determined by SYBRGreen based q-PCR using standard DNA as control.The residual host cell DNA was analyzed according to the standard curve.The developed method was verified by primer specifity, results accuracy and precision and used for determination of 3 batches of interferon substances. Results The minimum quantitative limit of residual host cell DNA by the developed method was 12 fg/μL, while the linear range was 12 fg/μL-120 ng/μL, with a correlation coefficient (r) of 0.998.The designed primers were specific to the DNA templates.The recovery rates of spiked samples with different DNA quantity were between 50%-200%.The residual host cell DNA determined by this method were not more than the limit, which were complied with the requirements for residual host cell DNA in Chinese Pharmacopeia ( volume III,2010 edition and 2015 edition) .Conclusion The wako DNA extractor kit could successfully solved the technical difficulties of sample pretreatment during residual DNA assay.The q-PCR method was simple, rapid and accurate for quantitation of residual host cell DNA in interferon substances.

Objective To develop a reversed phase high performance liquid chromatography ( RP￣HPLC) method determining the related substances and levidipine. Methods The Welchrom C18 column (250 mm×4.6 mm,5 μm) was used with Octadecylsilane bonded silica as a filler.The mobile phase consisted of methanol￣ethanol￣water (40:20:40),at the flow rate of 1 mL.min-1 in an isocratic elution,the temperature was at 45 ℃ , and the detective wavelength was 240 nm. Results Levidipine could be separated from all impurities and intermediates within the concentration range from151 to 604 ng.mL-1 , which had a satisfied linear relationship (r= 0.999 9) with a regression equation of Y= 0.049 9X+0.597 9.The LOQ of detection was 31.9 ng.mL-1 . Conclusion The developed method is specific,sensitive,easy and fast to operate,which is suitable for detecting levidipine and its related impurities.

Objective A high performance liquid chromatographic (HPLC) method was established for the determination of glycerol in propofol medium and long chain fat emulsion injection.MethodsThe chromatographic conditions were as follows: Kromasil 100-5-NH2 column(4.6×250mm,5μm) with the column temperature was 40℃,acetonitrile-water(8515)as mobile phase with flow rate of 1.0mL/min.Glycerol was detected by refractive index (RI) detector at 40℃.ResultsThe linear range of glycerol was 455.3916-2276.9580μg/mL(r=0.9999,n=7),the average recovery rate was 99.5%,RSD was 0.6%(n=9),the limit of detection(LOD) was 121ng and the limit of quantification(LOQ)was 364ng.ConclusionThe method was simple, rapid, strong specifity and accurate with good reproducibility, which is suitable for the content determination of glycerol in propofol medium and long chain fat emulsion injection.

Objective To study the correlation between the renin-angiotensin-aldosterone system angiotensinogen (AGT) gene M235T,angiotensin Ⅱ type 1 receptor (AGTR1) gene Al166C,aldosterone synthase (CYP11B2) gene -344C/T polymorphisms and large-artery atherosclerotic (LAA) stroke in a southern Chinese Han population.Methods Polymerase chain reaction and gene sequencing technology were used for the genotyping in patients with LAA and normal controls with AGT gene M235T,AGTR1 gene A1166C,and CYP11B2 gene - 344C/T polymorphisms in a southern Chinese Han population,and to determine the correlation between the 3 gene polymorphisms and LAA by binary logistic regression analysis.Results A total of 107 patients with LAA and 142 healthy controls were included in the study.The frequencies of the AGT gene 253TT genotype (66.36％ vs.50.70％,x2 =6.122,P =0.047) and T allele (79.44％ vs.70.07％ ％,x2 =5.581,P =0.018) in the LAA group were significantly higher than those in the control group.The frequencies of the AGTR1 gene 1166CC genotype (0％ vs.0％,x2 =1.494,P =0.222) and C allele (7.48％ vs.4.93％,x2 =1.399,P =0.237) in the LAA group were no significantly differences with those in the control group.The frequencies of the CYP11B2 gene - 344CC genotype (9.35％ vs.4.23％,x2 =3.603,P =0.165) and C allele (27.10％ vs.26.06％,x2 =0.069,P =0.793) in the LAA group were no significant differences with those in the control group.Binary logistic regression analysis showed that there was no significant correlation between the three gene polymorphisms and the simple LAA diseases.The frequencies of AGT gene 235TT genotype (68.00％ vs.41.90％,x2 =12.446,P =0.002) and T allele (79.33％ vs.64.76％,x2 =8.993,P =0.003) in the LAA patients complicated with hypertension were significantly higher than those in the normotensive control group.Logistic regression analysis showed that the odds ratio (OR) exposed to TT genotype was 2.153 (95％ confidence interval [CI] 0.789-5.872).The OR of T allele was 2.089 (95％ CI 1.285-3.396).Conclusions The AGT gene M235T polymorphism is not associated with the simple LAA in the southern Chinese Han population,but it may be associated with the risk of LAA complicated with hypertension;CYP11B2 gene -344C/T polymorphism and AGTR1 gene A1166C polymorphism are not associated with the onset of LAA in the southern Chinese Han population.

To investigate the freshness, high molecular weight substances, the determination of polypeptide, haemolysis and agglomeration, biological activity of Cervus and Cucumis polypeptide injection; to provide the direction for improving the quality of products for enterprises; furthermore, to provide reference for the revision of the quality standards of Cervus and Cucumis polypeptide injection. Firstly, we investigated the factors affecting the freshness of the injection, including biogenic amines, aflatoxins, the acid value and peroxide value of the melon seeds. The method of dansyl chloride pre-column derivatization-HPLC was used to determine the content of 8 biogenic amines in Cervus and Cucumis polypeptide injection. The method validation results showed good specificity, precision, linearity and recovery rates, which was suitable for the determination of biogenic amines in Cervus and Cucumis polypeptide injection. The results of sample determination showed that relatively higher concentrations of cadaverine were detected in the products from company B. The results of aflatoxins, acid value and peroxide value showed that the melon seeds from some companies had rancidity, mildew and other problems, indicating that the quality standards of multi-component biochemical drugs containing animal- and plant-derived components should be controlled in terms of freshness. Secondly, the methods for the determination of high molecular weight substances and polypeptides in the quality standard were improved. Tricine-SDS-PAGE electrophoresis was used instead of gel chromatography to determine the high molecular weight substances, which improved the accuracy of determination. The kits were used instead of folin-phenol for the determination of peptide content, which is easy to operate, specific and suitable for high-throughput sample determination. Finally, the haemolysis, agglomeration, and biological activity of Cervus and Cucumis polypeptide injection were studied. The results showed that no haemolysis and agglomeration were found in all samples, and the inhibitory effect of samples on THP-1 proliferation in vitro from different companies was different to some extent. In conclusion, the optimized quality standard is more suitable for the detection of Cervus and Cucumis polypeptide injection, and can lay the foundation for improving the safety of multi-component biochemical drugs.