1.Study on the preparation method for lower-sampling and large-scale purification of a wound-healing agent: fibronectin
Jianrong TAN ; Guomei REN ; Libing DAI ; Xuefang HUANG
Chinese Journal of Tissue Engineering Research 2005;9(47):167-169
BACKGROUND: Fibronectin serves not only as the supporter for cells,but also as an important intracellular linkage, possessing opsonic-like functions. It can promote the phagocytic function of mononucleophages and the repair in inflammation and trauma.OBJECTIVE: Type O plasma was freshly obtained from healthy males in search of the optimal preparation method for lower sampling, convenient and higher-yielding of fibronectin.DESIGN: Open experiment.SETTING: Institute of Traumatic Surgery, Fourth Affiliated Hospital of Guangzhou Red Cross Hospital, Jinan University.MATERIALS: This experiment was carried out in the Institute of Traumatic Surgery of Guangzhou Red Cross Hospital between July 2000 and July 2002. The major materials consisted of type O fresh plasma from healthy males, gelatin, sepharose 4B activated with cyanogen bromide,sephadex G-25, urea, and trishydroxymethylaminomethane.METHODS: Affinity column constituted by gelatin coupling with sepharose 4B activated with cyanogen bromide was used to purify fibronectin. ① Human plasma was added: 150 mL type O plasma was freshly obtained from healthy males and added into the column once by 25 mLat an interval of 20 minutes, the speed of flow was 3.5 mL/min. ② Removing mixed protein: the column was rinsed by Tris-sodium citrate equilibrium liquid (pH 7.5) until the absorbency of flow-out fluid decreased to < 0.02 at 280 nm. Again lmol/L NaCl containing Tris-sodium citrate was used to wash off other mixed proteins. ③ Collecting fibronectin: the column was rinsed by urea-Trispurge Fluid (3 mol/L) to collect fibronectin.④ Removing urea from fibronectin collection: fibronectin collection liquid was filtrated by Sephade G-25 to remove urea. ⑤Disinfection: 0.22 μmgermtight filter was used for disinfection.MAIN OUTCOME MEASURES: ①Results of sodium dodecyl benzene sulfonate-polyacrylamide gel electrophoresis. ② The influence of retention time on the yield of purified fibronectin. ③ The influence of plasma quantity on the yield of purified fibronectin.RESULTS: ① Results of sodium dodecyl benzene sulfonate-polyacrylamide gel electrophoresis: The density of separation gel and condensed gel was 7% and 3%, respectively; fibronectin was electrophoresized into single protein lane. ② The influence of retention time on the yield of purified fibronectin: The yield of fibronectin was (65.24±3.45) %, (74.77±4.05) %,(86.99±4.10) %, and (80.47±3.75) %, respectively, when the loading amount of fibronectin was 150 mL with non-retention time and retention time of 10, 20 and 30 minutes. ③ The influence of plasma quantity on the yield of purified fibronectin: The yield of fibronectin was (72.56±3.63) %,(77.61±3.14) %, (86.99±4.12) %, and (74.67±3.05) % when the column retention time was controlled at minute 20 with the loading amount of 100,130, 150 and 180 mL, respectively.CONCLUSION: In a given column volume of gelatin, the quantity of purified fibronectin was closely related with the plasma retention time in column and the total loading amount of plasma. As a result, the optimal loading amount of plasma was 150 mL, and the retention time was 20 minutes.The preparation method, herein, has been proved to require small amounts of plasma and yield large amounts of fibronectin.
2.Role of human plasma fibronectin in the repair of the refractory corneal epithelial defect
Guomei REN ; Libing DAI ; Huizhen YE ; Peihong LIANG ; Na LIANG ; Weizhong YANG ; Yetao CHEN ; Huiying XU ; Jianxin CHENG
Chinese Journal of Tissue Engineering Research 2005;9(2):216-217
BACKGROUND: Fibronectin(FN) plays the role of repair in the inflammation. There is no confirmed conclusion whether it can be applied to the refractory corneal epithelial defect.OBJECTIVE: To observe the repair effect of the human plasma FN for the refractory corneal epithelial defect.DESIGN: A controlled experimental study.SETTING: Guangzhou Red Cross Hospital, Guangzhou First People's Hospital, Guangzhou Children's Hospital, Guangzhou Hospital of Traditional Chinese Medicine and Guangzhou Second People's Hos pital.PARTICIPANTS: Totally 383 eyes with the refractory corneal epithelial defect were chosen, of which 309 were in the therapy group, and 74 in the control group.METHODS: The therapy group: Human plasma FN was administered by dropping it into the eyes once every two hours. The controlgroup: 10 g/L celacol M was administered by its dribbling into the eyes once every two hours. Weilesheng was taken orally in both groups, two pills once, three times per day. According to the state of illness, both groups received anti-bacterial or anti-viral treatment and reexamination was given every day or every other day after administration. 10 g/L fluorescein sodium was used to observe the changes of cornea.MAIN OUTCOSE MEASURES: The symptoms, results of staining using fluorescein sodium as well as the corneal epithelial healing of both groups.RESULTS: The symptoms, the results from staining using the fluorescein sodium and the corneal epithelial healing were used for the evaluation. In the therapy group, 309 eyes were followed up and the cure rate was 69.9%. The average therapeutic period was 6.5 days, while those of the control group were 58.1% and 8.7 days respectively. The difference in the curative effect between the two groups was significantly different( P<0.01 ).CONCLUSION: The application of FN for the refractory corneal epithelial defect displays a more significant effect than conventional treatment.
3.The EcoR V polymorphism of human monoamine oxidase A is not associated with idiopathic Parkinson's disease in a Shanghai Han population.
Huijun XIE ; Xiaohua WANG ; Yixin HAO ; Guomei TANG ; Ling XU ; Qihan WU ; Lishan CHEN ; Daming REN
Chinese Journal of Medical Genetics 2002;19(4):329-331
OBJECTIVETo explore the distribution of monoamine oxidase A (MOA-A) EcoRV polymorphism in Shanghai Han population and its possible role in the risk for Parkinson's disease(PD).
METHODSThe MAO-A gene EcoRV polymorphism was detected with PCR-RFLP method in 110 PD patients and 182 healthy controls, furthermore, statistical analysis was performed to investigate association between EcoR V polymorphism and PD onset.
RESULTS(1)Remarkable difference in MAO-A EcoR V polymorphic distribution has been observed between Shanghai Han population and that in North America. (2) Neither allelic frequency nor genotypic frequency in PD cases differs significantly from that in healthy controls regardless of data from male or female subclass.
CONCLUSIONThere may be racial difference in the distribution of the human MAO-A EcoR V (C/T) polymorphism, but the present research does not support the association between this variant and susceptibility to PD in Chinese Han population of Shanghai area.
Adult ; Aged ; Aged, 80 and over ; Alleles ; China ; DNA ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Monoamine Oxidase ; genetics ; Parkinson Disease ; enzymology ; genetics ; Polymorphism, Genetic