| Seed Cells Optimization and Post-transplant Regulation by Gonadotrophinin Tissue-Engineered Androgen-Secreting Tissue ConstructionBackgroundIn recent years, with the increase of congenital disease, environmental pollution, socialpressure and ageing populations, the morbidity of hypoandrogenism increases year by year,and testes loss caused by trauma or surgical procedure for tumor treatment was not rare inclinical practice.However, until now there was no safe and effective therapy for treating this kind ofdisease. With the development of tissue engineering technology, construction oftissue-engineered androgen-secreting tissue showed exceptional advantage in treatinghypoandrogenism or testes loss. But, the limited source of seed cells had become thebottlenecks of this treatment and study. In our previous study, we found that the Leydig cell(LC) could be purified by using the different attachment rates of the cells digested from thetestes. Although, this new method had solved the problem about source of seed cellspreliminarily, there was no quantitative compare in the yield, purity and secreting functionbetween the new and traditional method. In addition, the optimal time of adherent culture,which results in the best yield and purity, had not been validated experimentally. Because ofthe regulator role of the Sertoli cell (SC) on LC function, the combined cells obtained fromthe entire testes had been proven better than the LC as the seed cell of the androgen-secretingtissue. But the appropriate ratio between the two cells for the treatment of cell transplantationor construction of androgen-secreting tissue wasn’t clear yet. In the digestion procedure of thenew method for the LC purifying, the residual seminiferous tubules kept integrated instructure offered a possibility to isolate the SC further. Moreover, the androgen-secretingtissue could maintain a certain level of testosterone secreting in18weeks after theimplantation. But, there has been some question as to whether or not the testosteronesecreting could be regulated by the hypothalamic-pituitary and then accord with the inherentphysiology rhythm.Objective At first, we aimed to prove the practicability of the new method (different attachmentrates) by comparing it with the traditional method (density gradient centrifugation) in theyield and purity of the isolated LC, and confirm the optimal time of adherent culture.Secondly, we planned to isolate the SC from the residual seminiferous tubules after thedigestion for LC and co-cultivate the two cells in several ratios to detect the differenttestosterone level in supernatant. Thirdly, we constructed the androgen-secreting tissue usingthe LC and SC isolated separately as the seed cells, and implanted the tissue into the castratedrats. By injection of the exogenous gonadotropin releasing hormone and detection of serumtestosterone and luteinizing hormone (LH), it could be validated whether or not thetestosterone secreting could be regulated by the hypothalamic-pituitary-gonad axis(HPG-axis). This study tried to optimize the seed cells for the androgen-secreting tissueconstruction and establish a method to estimate the function after implantation.Methods1. Isolation of rat LC by using different attachment rates and density gradientcentrifugation:After decapsulation, digesting, filtering, centrifuging and resuspending, four-fifths of theinterstitial cell suspensions were adherent cultured with the first medium change at4differenttime points, while the rest used the traditional method as a comparison. By comparing theyield and purity of the isolated LCs between4different time points, an optimal time wasdetermined and represented the new method. A series of identification and function assaywere performed to compare the LCs isolated by using the new and traditional method.2. Isolation and purifying of rat SC:By using the mixed enzymes, the SCs were isolated from the residual seminiferous tubulesimmediately after the digestion for LC, and underwent a series of identification and detectionfor yield and purity. After72hours culture, the SCs were co-cultured with the LCs in differentratios (1:1,1:4,1:9and0:1) to detect the difference in the testosterone level of supernatant.3. Construction of the androgen-secreting tissue using the LC and SC isolatedseparately as the seed cells:The LCs and SCs isolated separately using above-mentioned method were combined inprimary ratio and seeded onto the pre-formed biodegradable scaffold. After24hour vitro culture, the cell-scaffold composites were implanted into the gastrocolic omentum of the rats,which had been castrated for4weeks. The testosterone and luteinizing hormone in serum ofthe implantation and castrated rats were detected every week.4. Interventions of LHRH to the normal, castrated and implantation rats:The exogenous gonadotropin releasing hormone (LHRH) was injected subcutaneously to therats of normal, castrated and implantation group everyday for a week. Meanwhile a group ofimplantation rats were injected subcutaneously by the normal saline. The serum testosteroneand LH of each group were detected after the injection for1and7days to show theintervention effects in short-term and long-term injection respectively. Furthermore, a seriesof feeding and blood collection were performed on a group of the implantation rats accordingto circadian rhythms strictly to analyze the circadian difference in the serum testosterone andLH.ResultsPart Comparison between different attachment rates and density gradientcentrifugationTwo hours was the optimal duration of adherent culture for LC isolation and resulted in theaverage yield of (2.04±0.26)×106cells per gram (wet weight of the testis) with the averagepurity of (75.22±3.14)%, both of which were superior to the LCs isolated by using densitygradient centrifugation. Besides, testosterone secreting function of the LCs in new methodwas also better those in traditional method due to the preservation of the Leydig stem cells.Considering the simple procedures, which were efficient and easy to repeat with minimaldamage, different attachment rates would be of great value for the researches about cellulartherapy and reconstruction of androgen-secreting tissue.Part Isolation of LCs and SCs step by step and vitro culture in different ratioBy using the mingled enzymes consisting of trypsin, collagenase, hyaluronidase and DNase,the SCs were isolated from the residual seminiferous tubules immediately after the digestionfor LC. The average yield and livability of SC were (3.5±0.62)×106cells per gram (wetweight of the testis) and about95%respectively. The testosterone level of supernatantmaintained for the longest time when the SCs were co-cultured with the LCs in the ratios of Part Regulation of LHRH on the androgen-secreting tissue constructed by using thecombined cells as seed cellsBoth of the LCs and SCs isolated step by step showed good biocompatibility with the scaffold.The construction of androgen-secreting tissue maintained a certain volume and level ofsecreting function in3months after implantation. The secreting couldn’t only increase withthe up-regulation of hypothalamic-pituitary, but also give negative feedback to the former invivo.ConclusionThis study confirmed that it’s feasible to obtain the Leydig lineage cells efficiently by usingthe different attachment rates, and the new method was superior to the traditional one in theyield, purity and testosterone secreting function of the LCs. By using the mixed enzymes, theSCs could be isolated from the residual seminiferous tubules immediately after the digestionfor LC. The androgen-secreting tissue constructed by using the combined cells as the seedcells showed good secreting function, which could increase with the up-regulation ofhypothalamic-pituitary. In conclusion, this study optimized the ratio of the2kinds of seedcells preliminarily, and established a method to estimate the function after implantation for theandrogen-secreting tissue construction. |
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