Study on the preparation method for lower-sampling and large-scale purification of a wound-healing agent: fibronectin
- VernacularTitle:少加样高收效制备创伤修复剂纤维粘连蛋白的方法
- Author:
Jianrong TAN
;
Guomei REN
;
Libing DAI
;
Xuefang HUANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2005;9(47):167-169
- CountryChina
- Language:Chinese
-
Abstract:
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.
