骨髓基质细胞条件培养液中神经活性物质的提取与纯化

目的分离纯化骨髓基质细胞(bone marrow strolnal cells，BMSCs)条件培养液以获得某些神经活性物质。方法分离培养小鼠骨髓基质细胞并收集其培养液，超滤浓缩后采用Sephadex G-100层析，高效液相色谱分析(HPLC)和十二烷基硫酸钠-聚丙烯酰胺凝胶不连续电泳(SDS-PAGE)等技术分离其培养液中的蛋白组分并利用细胞培养技术验证其神经活性作用。结果骨髓基质细胞培养液经Sephadex G-100层析及HPLC分析，证实Ⅲ峰中的A峰和B峰对神经细胞生长有明显的促进作用，其相对分子质量分别为18000和14000。结论骨髓基质细胞条件培养液中相对分子质量为18000和14000两组分对神经元有营养活性。     

雪旺细胞胞浆神经营养蛋白的分离纯化和理化性质测定

目的：分离纯化雪旺细胞胞浆神经营养蛋白并测定其等电点。方法：培养收集新生１－３天ＳＤ乳鼠四肢神经雪旺细胞，超声粉碎，超速离心，取上清胞浆成份超滤浓缩，收集分子量＞１０ＫＤａ浓缩蛋白液，通过ＤＥＡＥ－Ｓｅｐｈａｃｅｌ离子交换层析，ＳｅｐｈａｄｅｘＧ－１００凝胶过滤层析和Ｄｉｏｌ－１５０高压液相分子筛层析进行分离纯化，等电聚焦电泳测定纯化的活性蛋白质等电点和ＳＤＳ－聚丙烯酰胺凝胶电泳测定其亚基分子量。结果：成功地分离纯化出一种活性蛋白质，分子量约５８ＫＤａ，等电点４．５５，结论：联合应用超滤浓缩、离子交换层析、凝胶过滤层析和高压液相能较理想地分离纯化雪旺细胞胞浆蛋白     

雪旺细胞浆神经营养蛋白高效液相分离纯化及其生物活性研究

目的　分离纯化雪旺细胞浆神经营养蛋白并研究其生物活性。方法　取 30 0只出生后 1～ 3天SD大鼠双侧坐骨神经 ,纯化培养 ,收集雪旺细胞 ,超声粉碎 ,超速离心 ,不同孔径分子筛滤膜 (PM10、30、5 0 )超滤浓缩 ,收集分子量 10～ 30 ku,30～ 5 0 ku和 >5 0 ku三种组份胞浆蛋白浓缩液 ,分别加入体外无血清培养的 E14SD胎鼠脊髓运动神经元培养液中 ,用 MTT酶标法检测证实 10～ 30 ku和 >5 0 ku两组份蛋白有神经营养活性。再通过Diol- 15 0高效液相分子层析进一步从雪旺细胞胞浆中纯化分离出分子量为 2 6 ku和 5 8ku两种胞浆蛋白 ,并对其神经生物活性进行研究。结果　 2 6 ku和 5 8ku雪旺细胞胞浆蛋白能维持体外无血清培养的脊髓运动神经元存活 ,其最佳生物活性浓度为 2 0 ng/孔。结论　雪旺细胞胞浆内含有分子量为 2 6 ku和 5 8ku的运动神经营养蛋白     

雪旺细胞分泌神经营养蛋白的提纯

目的：寻找出提纯神经营养蛋白的实用而有效的方法。方法：收集ＳＤ乳鼠周围神经的雪旺细胞无血清条件培养液，离心，将上清液超滤浓缩（ＰＭ１０）收集分子量大于１０ＫＤａ浓缩蛋白液，经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤获得三个蛋白峰，经生物活性检测，证明洗脱Ⅱ峰液生物活性最大。将冼脱Ⅱ峰液再经ＰＭ１０超滤膜浓缩收集浓缩蛋白液，分别用ＴＨＫ２４、ＴＴＫ２４微型过滤器去除大于１００ＫＤａ和小于３０ＫＤａ蛋白，过分子筛高效液相色谱柱。结果：得到４个主要蛋白峰，前三峰蛋白分子量均超过１５０ＫＤａ，第四峰分子量为６７ＫＤａ，经ＳＤＳ－ＰＡＧＥ和银染色法进一步证实该蛋白分子量为６７ＫＤａ，基本为单一蛋白带。结论：Ｓｅｐｈａｄｅｘ凝胶过滤、多次超滤和分子筛高效液相色谱技术综合应用是纯化雪旺细胞源神经营养蛋白较好的方法。     

人骨肉瘤原代细胞条件培养基中骨形态蛋白(BMP)的实验研究

目的:探索从人骨肉瘤细胞条件培养基中提取骨形态蛋白(BMP)的方法,并测定其生物学活性.方法:收集人骨肉瘤细胞条件培养基,通过浓缩、透析,SephcrylS-100凝胶层析纯化,BMP单克隆抗体鉴定所需洗脱峰,SDS-PAGE测定分子量,小鼠肌袋实验检测其骨诱导活性.结果:BMP单抗鉴定所提蛋白为BMP,SDS-PAGE显示分子量约为21kD,能够在小鼠肌肉内产生异位骨化.结论:人骨肉瘤细胞条件培养基中含有BMP,分离后具有良好的生物学活性,而骨肉瘤细胞可以在体外长期培养生长,为BMP的大量提取、临床应用提供一个有益的方法.     

人骨肉瘤原代细胞条件培养基中骨形态蛋白(BMP)的实验研究

目的：探索从人骨肉瘤细胞条件培养基中提取骨形态蛋白(BMP)的方法，并测定其生物学活性。方法：收集人骨肉瘤细胞条件培养基，通过浓缩、透析，SephcryIS-100凝胶层析纯化，BMP单克隆抗体鉴定所需洗脱峰，SDS-PAGE测定分子量，小鼠肌袋实验检测其骨诱导活性。结果：BMP单抗鉴定所提蛋白为BMP，SDS—PAGE显示分子量约为21kD，能够在小鼠肌肉内产生异位骨化。结论：人骨肉瘤细胞条件培养基中含有BMP，分离后具有良好的生物学活性，而骨肉瘤细胞可以在体外长期培养生长，为BMP的大量提取、临床应用提供一个有益的方法。     

雪旺氏细胞分泌蛋白质及其神经营养活性物质的生化分析

成年大鼠瓦勒氏变性神经来源的雪旺氏细胞（ＳＣ），经体外无血清分离培养，收集ＳＣ无血清条件培养液，用ＳＤＳ－ＰＡＧＥ分析其超滤浓缩液（分子量大于１０ＫＤａ）中ＳＣ分泌蛋白，发现其含有１４条蛋白带，分子量从大于９４ＫＤａ至小于３４ＫＤａ。用免疫印迹技术、抗２．５Ｓ神经生长因子（ＮＧＦ）抗体鉴定证实ＳＣ分泌蛋白中含有ＮＧＦ。用Ｄｉｓｃ－ＰＡＧＥ（４℃）分离ＳＣ分泌蛋白带，分别经电洗脱、浓缩收集１０份蛋白区带，结合有髓腹侧运动神经元细胞培养、ＮＴＦｓ生物学活性鉴定，初步发现其中一组蛋白区带（Ｂ＋）含运动性神经营养因子（ＮＴＦ）活性，其分子量在６５ＫＤａ至３４ＫＤａ。     

雪旺细胞浆神经营养蛋白高效液相分离纯化及其生物活 …

目的 分离纯化雪旺细胞浆神经营养蛋白并研究其生物活性。方法 取３００只出生后１～３天ＳＤ大鼠双侧坐骨神经,纯化培养,收集雪旺细胞,超声粉碎超速离心,不同孔径分子筛滤膜（ＰＭ１０、３０、５０）超滤浓缩,收集分子量１０～３０ｋｕ,３０～５０ｋｕ、〉５０ｋｕ三种组份乐蛋白浓缩液,分别加入体外无血清培养的Ｅ１４ＳＤ胎鼠脊髓运动神经元培养液中,用ＭＴＴ酶标法检测证实１０～３０ｋｕ,〉５０ｋｕ两组份蛋白有神经     

胆汁泡蛋白成核活性及其氨基酸序列分析的研究

分离胆汁泡蛋白,研究其成核活性和末端氨基酸序列。方法:应用超速离心法分离胆石症患者胆汁泡,运用SDS-PAGE电泳分离其中胆汁泡蛋白,并经HPLC系统纯化,研究其成核活性和N-端氨基酸序列。结果:分离到一种分子量为33.5kd的胆汁泡蛋白,其促成核活性为0.30,N-端氨基酸具有较多的极性氨基酸(天冬酰、苏、丝和谷氨酰等)。结论:33.5kd胆汁泡蛋白有明显的促成核活性,其N-端极性氨基酸可能参与促成核机制。     

人脐血源和骨髓源间充质干细胞神经前体细胞分化的研究

目的 对比研究人脐血和骨髓来源的间充质干细胞(mesenchymal stem cells,MSCs)体外的分离、培养和生物学特性,并观察其分化潜能和形态学变化,为组织工程选取种子细胞提供实验依据.方法 Ficoll密度梯度离心结合贴壁培养法分别分离纯化成人骨髓和脐血源MSCs,体外培养和连续传代,并在含有2%B27的Neurobasal培养基中添加碱性成纤维细胞生长因子、表皮生长因子,将获得的MSCs向神经干细胞定向诱导,利用倒置显微镜连续观察细胞培养、传代和向神经细胞表型转化过程的形态学变化;采用免疫组化和荧光免疫组化法对诱导后细胞进行鉴定.结果 原代分离的骨髓间充质干细胞(BMSCs)在接种后48 h贴壁,7 d细胞呈长梭形,有一定的方向性,并达到90%融合;而脐血间充质干细胞(UMSCs)48 h后贴壁,似乎贴的不牢,持续14d才能形成小丛、小簇、小集落,21 d排列才有一定的方向性.培养基添加神经营养因子诱导后的细胞呈现典型的神经前体细胞样表型,免疫组化和免疫荧光结果显示,诱导后的细胞能特异性表达神经元特异性标志β微管蛋白(β-tubulin)和星形胶质细胞特异性标志神经胶质相关蛋白(GFAP).结论 人脐血和骨髓中含MSCs,且具备其基本恃征,体外培养UMSCs生长速度比BMSCs缓慢10 d～15 d左右,传代以后的各组细胞生长速度与形态无明显差异.骨髓和脐血来源的MSCs在体外可定向诱导分化为神经细胞.     

Studies on low Molecular Weight Follicle Stimulating Hormone Receptor Binding Inhibitor (FSH-RBI) from Ovine Testis

Follicle stimulating hormone receptor binding inhibitor (FSH-RBI) has been isolated from the aqueous extracts of ovine testis using Sephadex column chromatography. Sephadex G-75 fraction IV was found to inhibit the binding of (125I) FSH to rat testis receptors. Further purification of Sephadex G-75 fraction on Sephadex G-25 column gave three fractions (I-III). The maximum inhibitory activity to inhibit (125I) FSH binding to rat testis receptor was associated with fraction III only. FSH-RBI exerted a greater inhibitory effect on the formation of the hormone-receptor complex rather than on the dissociation of the preformed complex. FSH-RBI did not inhibit the binding of (125I) LH to rat testis receptors. A significant decrease in the mouse ovarian weight was observed when FSH-RBI was injected to hCG-primed female mice. The molecular weight determination studies show that molecular weight of FSH-RBI is approximately 1400 Daltons.     

Secretion of insulin-like growth factor I and its binding proteins by collecting duct cells.

Insulin-like growth factor I (IGF-I) has been found in the kidney, particularly in the collecting duct in the rat. Since cultured rabbit collecting duct cells constitute a convenient system for in vitro studies, we have examined whether these cells secrete IGF-I. Culture medium conditioned by collecting duct cells was concentrated by reverse phase chromatography and applied to a Sephadex G100 column equilibrated in a denaturing buffer. Two major species with apparent molecular weights of 7.5 and greater than 25 kilodaltons (kD) were identified by IGF-I RIA. A smaller amount of 10 kD species was also observed. Further characterization of 7.5 kD IGF-I immunoreactive species by reverse phase HPLC showed that it eluted in a single peak. To determine whether the higher molecular weight species possessed IGF-I binding activity, appropriate fractions were desalted, incubated with [125I]IGF-I (thr59) for two hours at 30 degrees C and applied to a Sephadex G100 column equilibrated in a non-dissociating buffer. The major peak of radioactivity was confined to a high molecular weight region; there was no radioactivity in the fractions corresponding to 7.5 kD. Western ligand analysis of unreduced conditioned medium identified two IGF-I binding species of 25 and 30 kD, similar in size to species observed in normal rabbit serum. 125I-IGF-I binding as assessed in a charcoal adsorption assay could be displaced by IGF-I and IGF-II but not by insulin. Further characterization of the 10 kD peak of IGF-I immunoreactivity indicated that it did not possess IGF-I binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enterolobin, a hemolytic protein from Enterolobium contortisiliquum seeds (Leguminosae--Mimosoideae). Purification and characterization

Hemolytic and phospholipase D activities were found in the saline extract of Enterolobium contortisiliquum seeds. The hemolytic activity is due to a protein which was named enterolobin. This protein was highly purified by extraction with 0.15 M NaCl, precipitation with ammonium sulphate from 0 to 33% of saturation, batch separation by adsorption on DEAE-cellulose and gel filtration chromatography on Sephadex G-100 or G-150. In the batch separation the fraction showing hemolytic activity was not adsorbed by the resin while the fraction with phospholipase activity was. In this manner it was shown that those two activities were due to different proteins. Mouse erythrocytes were less susceptible to hemolysis by enterolobin than human and rabbit erythrocytes. The hemolytic activity was rapidly lost at or above 55 degrees C and in extreme acid (1.6) and basic (10.8) pHs. The following characteristics of purified enterolobin were determined: molecular weights of 55.000 D (by SDS-PAGE), 59.800 D (by gel filtration) and 51.300 D (by HPLC); pI = 7.0; Gln as the N-terminal amino acid residue; high levels of Asp(Asx), Glu(Glx), Ser and Thr residues and low levels of Cys and Met residues. Similarities were noticed between enterolobin and crotin, a hemolytic protein of Croton tiglium seeds.     

Presence of Luteinizing Hormone Receptor Binding Inhibitor (LH-RBI) in Ovine Testis

Luteinizing hormone receptor binding inhibitor (LH-RBI has been isolated from the aqueous extracts of ram testis using Sephadex column chromatography. Sephadex G-75 fraction I was found to inhibit the binding of I125 LH to rat testis receptors. Further purification of Sephadex G-75-I fraction on G-200 column gave four fractions (I-IV), the maximum inhibitory activity to inhibit I125 LH binding to rat testis receptor was associated with the fraction I only. Fraction II gave marginal inhibition only, whereas fractions III and IV did not have any inhibitory effect on I125 LH binding to rat testis receptors. Our findings suggest that LH-RBI isolated from ram testis is a protein having molecular weight more than 10,000 Daltons.     

Dihydrotestosterone receptors in the human prostate: characterization by chromatography, electrophoresis, and isoelectric focusing

The human prostate androgen receptor was characterized by Sephadex G-100 chromatography, acrylamide-DATD gel electrophoresis, and isoelectric focusing. A KCl nuclear extract was prepared and incubated in the presence of tritiated DHT or R1881 at 4 degrees C for 18 h. In the absence of proteolytic enzyme inhibitors, a fraction of the hormone receptor complex was excluded from the G-100 column, whereas another fraction migrated with proteins having apparent molecular weights in the area of 34,000 daltons. The complex migrated on gel electrophoresis only after mild trypsinization. As for the isoelectric focusing, the nuclear complex showed two forms specifically bound to [3H]androgens with pI in the area of 4.7 and 7.9, respectively.     

Early detection of degraded A14-125I-insulin in human fibroblasts by the use of high performance liquid chromatography

We studied the metabolism of A14-125I-insulin in intact human fibroblasts using high performance liquid chromatography (HPLC) to detect and separate its early degradation products. The high resolving power of HPLC enabled us to separate what has been considered "intact insulin" by Sephadex G-50 chromatography or TCA precipitability into two additional peaks that had decreased biochemical properties with respect to immunoprecipitability and receptor binding but not decreased TCA precipitability. We conclude that human fibroblast is capable of metabolizing insulin within 2 min at 37 degrees C into intermediate molecules that can be detected by HPLC but not by TCA precipitability or molecular sieve chromatography.     

Purification and characterization of guinea pig lymphotoxin produced by lymph node cells stimulated by phytohemagglutinin.

Guinea pig lymphotoxin (LT) produced by stimulation of lymph node cells with Phaseolus vulgaris phytohemagglutinin was purified approximately 1,000-fold in specific activity by ammonium sulfate fractionation, DEAE-Sephadex column chromatography, and gel filtration on Sephadex G-100. The properties of LT tested at the various stages of purification revealed that the LT was a heat-sensitive, protease-sensitive proteinaceous substance, and its approximate molecular weight was estimated to be 50,000 by means of gel filtration. The most purified fraction (Fr. D) was found to be devoid of the activities of migration inhibitory and mitogenic factors, clearly indicating that these activities are mediated by different substances.