目的优化重组人角质化细胞生长因子-2(Recombinant human keratinocyte growth factor-2,rhKGF-2)基因工程菌的发酵条件,并对rhKGF-2进行纯化及活性检测。方法优化rhKGF-2基因工程菌接种量、装液量、培养基初始pH值、IPTG浓度、诱导时...目的优化重组人角质化细胞生长因子-2(Recombinant human keratinocyte growth factor-2,rhKGF-2)基因工程菌的发酵条件,并对rhKGF-2进行纯化及活性检测。方法优化rhKGF-2基因工程菌接种量、装液量、培养基初始pH值、IPTG浓度、诱导时机和诱导时间等发酵条件;rhKGF-2经离子交换层析和肝素亲和层析后,采用MTT法检测其对NIH-3T3细胞增殖的影响。结果 rhKGF-2基因工程菌的最佳发酵条件为:接种量2.0%,装液量50ml/250ml三角瓶,培养基初始pH值6.5～7.0,IPTG浓度0.10mmol/L,菌体处于对数生长初期时(A600=0.8～1.0)开始诱导,诱导时间4h;纯化的rhKGF-2经HPLC检测纯度达95.0%以上;在1～100μg/ml浓度范围内,rhKGF-2对NIH-3T3细胞的增殖具有促进作用。结论优化了rhKGF-2基因工程菌的发酵条件,获得了纯度较高、具有活性的rhKGF-2蛋白,为其工业化生产奠定了基础。展开更多
To investigate the proliferative ef fect of keratinocyte growth factor (KGF 2) on human adult keratinocytes. Methods: The standard medium was keratinocyte growth medium wit hout bovine pituitary extract (BPE), hydroco...To investigate the proliferative ef fect of keratinocyte growth factor (KGF 2) on human adult keratinocytes. Methods: The standard medium was keratinocyte growth medium wit hout bovine pituitary extract (BPE), hydrocortisone or epidermal growth factor ( EGF). Keratinocytes from a 48 year old subject were cultured and seeded on dis hes with standard medium of EGF in cell density of 2×10 4/32 mm 2. After 24 hours, the medium was replaced by the standard medium with 0, 4, 16, 125 and 50 0 ng/ml KGF 2, respectively. The standard medium with EGF was used as the posit ive control and the standard medium without EGF or KGF 2 was used as the negati ve controls. The growth of keratinocytes was monitored by 3 (4,5 dimethythiazo l 2 yl) 2,5 dipheyl tetrazolium bromide (MTT) assay and by photographs on day s 3, 5 and 7, respectively. Results: KGF 2 in concentrations of 4 500 ng/ml showed a sign ificant proliferative effect on days 5 and 7 as compared with that of the negati ve controls (P< 0.01 ). On day 3 the cells were prolifer ated to 1.5 2.5 fold, on day 5 to 3 5 fold and on day 7 to 3 12 fo ld in KGF 2 medium as that of the negative controls. The optimal response occur red when the concentration of KGF 2 was 125 ng/ml on day 7. Cell proliferation was also consistently higher in all KGF 2 concentrations as compared with that of the positive controls. Conclusions: KGF 2 has significant effects on the proliferatio n of adult keratinocytes, which are more effective than that of EGF. This study supports KGF 2 can improve the healing of chronic wounds in adults in clinic.展开更多
文摘To investigate the proliferative ef fect of keratinocyte growth factor (KGF 2) on human adult keratinocytes. Methods: The standard medium was keratinocyte growth medium wit hout bovine pituitary extract (BPE), hydrocortisone or epidermal growth factor ( EGF). Keratinocytes from a 48 year old subject were cultured and seeded on dis hes with standard medium of EGF in cell density of 2×10 4/32 mm 2. After 24 hours, the medium was replaced by the standard medium with 0, 4, 16, 125 and 50 0 ng/ml KGF 2, respectively. The standard medium with EGF was used as the posit ive control and the standard medium without EGF or KGF 2 was used as the negati ve controls. The growth of keratinocytes was monitored by 3 (4,5 dimethythiazo l 2 yl) 2,5 dipheyl tetrazolium bromide (MTT) assay and by photographs on day s 3, 5 and 7, respectively. Results: KGF 2 in concentrations of 4 500 ng/ml showed a sign ificant proliferative effect on days 5 and 7 as compared with that of the negati ve controls (P< 0.01 ). On day 3 the cells were prolifer ated to 1.5 2.5 fold, on day 5 to 3 5 fold and on day 7 to 3 12 fo ld in KGF 2 medium as that of the negative controls. The optimal response occur red when the concentration of KGF 2 was 125 ng/ml on day 7. Cell proliferation was also consistently higher in all KGF 2 concentrations as compared with that of the positive controls. Conclusions: KGF 2 has significant effects on the proliferatio n of adult keratinocytes, which are more effective than that of EGF. This study supports KGF 2 can improve the healing of chronic wounds in adults in clinic.
