| ObjectiveA series of evidence show that the level of free hyaluronic acid (HA) in the blood was higher than normal level was found when investigating tumor, hepatopathy, ophthalmology disease, skeleton disease etc. HA ,which was bound with cartilage link protein(CLP) in form of covalent bond compound , was mainly distributed in the intercellular substance. The compound of HA-CLP was separated by a certain concentration of guanidine hydrochloride solution,while the trace-study of piglet cartilage link protein (p-CLP) had not been reported currently. In this study, we conducted experiments to investigate the biological characteristics of p-CLP, to provide an experimental evidence for the lung tumor imaging and the early diagnosis of lung tumor .Four exploration were carried out for the studies.1. The separation and extraction of p-CLP. 2. The binding characteristics between p-CLP and HA. 3. The distribution of HA in lung tumor of nude mice. 4. The distribution of p-CLP in nude mice xenograft model of lung tumor. Methods1. Distribution of HA in lung tumor of nude mice The BALB/C-nunu nude mice that was inoculated by A549 cell was kept 3,4,5,6 weeks respectively.HA which distributed in nude mice xenograft of lung tumor was surveyed and evaluated by Streptavidin-Perosidase (SP) method of immunohistochemistry staining.2. The separation, purification and identification of p-CLP p-CLP was separated and purified by affinity chromatography from piglet costal cartilage. The Sepharose 4B was activated by the epichlorohydrin method, coupled with AH immediately and HA was coupled with AH-Sepharose4B successively. Furthermore, p-CLP was obtained from the semifinished product of p-CLP by a series of methods as follow: homogenate, enzymatic hydrolysis, concentration, and elution chromatography. The p-CLP was identified by SDS-PAGE gel electrophoresis analysis, L-8800 type -amino acid analysis instrument analyzing he amino acid content of p-CLP, and in-vitro radioactive competitive binding assay evaluating the affinity between p-CLP and HA3. The iodine labeling and trace analysis of p-CLP p-CLP was labeled with Na125I by choramine-T oxidation method .The best condition of labeling was explored to prepare high-purity radioactive probe (125I-p-CLP).The trace experiment of p-CLP was conducted as following methods using 125I-p-CLP, At first, 125I-p-CLP was injected into the nude mice of lung tumor xenograft through tail vein , then the nude was sacrificed at different moment respectively (5min,20min,30min,45min,1h,1.5h, 3h,5h,7h after injection) and the essential organs and blood was taken out and their radioactivity was measured to acquire the parameters of p-CLP's distribution and metabolism in nude mice.Results1. The positive rate of HA immunohistochemistry staining was 93%, optical density(OD) of positive organization after 3-6 weeks were 150.78±10.25,160.13±8.54,176.83±13.46,185.95±13.61, respectively, the control group was 37.43±5.17 .There were statistically significance among the groups (P<0.05). The immunohistochmistry of HA showed that HA expression was stronger and stronger as the extension of tumor growth time, and there was a up-regulation of HA expression in extracellular matrix in tumor cells and HA expression of tumor tissue was much richer than normal tissue's.2. The results of ultraviolet scanning showed that the map of p-CLP extracted using affinity chromatography had only a peak. The yield rate of CLP was 2.21%, SDS-PAGE gel electrophoresis showed two bands of p-CLP｛(44.36±2.90)KD, (71.58±4.13)KD respectively｝, amino acid assay showed that the content of glutamic acid(Glu) was the highest(2.26mg/20mg) and the content of Tyrosine(Tyr) was 0.91mg/20mg, in-vitro radioactive competitive binding assay showed that the affinity constant between p-CLP and HA was(6.30±1.73)×109M-13. Labeling rate of p-CLP had statistically significance among these groups (P<0.05) that radioactivities of 125iodine were 0.925MBq, 1.85MBq, 3.7MBq, 7.4MBq respectively, and no statistically significance (P>0.05) among 7.4MBq, 11.1MBq, 14.8MBq groups , and Labeling rate of p-CLP had statistically significant difference from other groups (P<0. 05) when the concentrations of choramine-T were 0.312mg/ml, 0.625mg/ml , no statistically significant(P>0.05) among other groups. Labeled time had little effect to the labeling rate (P>0.05). And radioactivity of 125iodine, concentrations of choramine-T and labeling time also had little effect on radiochemical purity (P>0.05). The bioactive of 125I-p-CLP was the highest when the concentrations of choramine- T was 0.625mg/ml, labeling time was 1 min, respectively .Radioactive trace experiments showed that p-CLP had the peak at 20min and the percentage of p-CLP of tumor was elevatory gradually with extension of time.Conclusions1. The expression of HA was an increased tendency in tumor tissues, there was the up-regulation of HA expression in extracellular matrix in tumor cells, and HA expression was stronger with the extension of tumor growth.2.The best Labeling condition of p-CLP was that radioactivities of 125I was 7.4MBq, concentrations of choramine-T was 1.25mg/ml, quality of p-CLP was 0.5mg, reaction volume was 100ul ,and reaction time was 1min.3. The trace experiments showed that p-CLP was widely distributed in murine body early. There was a specific-binding concentration of p-CLP in the stromal of lung tumor. |
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