公山羊Protamine 1 mRNA表达特性及与精液品质相关性的研究

旨在研究雄性山羊主要繁殖器官中PRM1mRNA表达特性,分析精子PRM1mRNA表达与精液品质参数的相关性。本研究通过荧光定量PCR技术分析PRM1mRNA表达规律,利用免疫组化技术对睾丸中PRM1蛋白进行定位,并应用GraphPad Prism 5软件分析精子PRM1mRNA表达量与精液品质指标的相关性。结果显示,PRM1mRNA在睾丸中高度表达,其次是附睾,睾丸中PRM1mRNA表达量是附睾头的247倍,附睾头显著高于附睾体和尾(P<0.05),在剩余其他组织中微量表达。在周岁之前,睾丸中PRM1mRNA表达量随着月龄增加而增加,12月龄的表达量显著高于9月龄的表达量(P<0.05),9月龄表达量显著高于其他低月龄的表达量(P<0.05)。精子PRM1mRNA的表达量与精子活力(R2=0.586 0,P=0.000 5)、精子密度(R2=0.442 2,P=0.004 9)呈显著正相关。山羊PRM1在长形精子细胞和精子细胞中表达,睾丸其他生精细胞及间质细胞中无表达。山羊PRM1mRNA表达具有时空表达特性,PRM1mRNA表达量可以作为评价公羊繁殖力的指标。

Analysis of the methylation pattern of six gene promoters in sperm of men with abnormal protamination

It has recently been shown that alteration of the methylation pattern of imprinted genes is associated with different types of male infertility. The objective of our study was to investigate the methylation pattern of selected gene promoters in sperm of patients with abnormal protamine replacement. The promoters of OCT4, SOX2, NANOG, HOXC11, miR-17and CREMwere analyzed using bisulfite sequencing and the percentage of DNA methylation was compared between patients with an abnormal protamine l/protamine 2 （P1/ P2） ratio and normozoospermic controls. No significant quantitative differences were found between groups of patients with either an abnormally high or low P1/P2 ratio compared to normal controls. However, two individual samples from infertile subjects （2/20, 10%） showed an altered methylation pattern for the CREMgene promoter that was not found in control samples. These two samples had a significantly higher （P〈0.05） promoter methylation （5.58 and 4.23%, respectively） compared to the control group （0.46%）. In conclusion, in our pilot study, extreme methylations defects were not seen broadly in severely infertile men. However, two patients exhibited altered methylation of the CREMgene, which may be either causative or a result of abnormal protmaine replacement.

Characterization of nucleohistone and nucleoprotamine components in the mature human sperm nucleus

Aim： To simultaneously determine the localization of histones and protamines within human sperm nuclei. Methods： Immunofluorescence of the core histones and protamines and fluorescence in situ hybridization of the telomere region of chromosome 16 was assessed in decondensed human sperm nuclei. Results： Immunofluorescent localization of histones, protamine 1 （PRM1） and protamine 2 （PRM2） along with fluorescence in situ hybridization localization of chromosome 16 telomeric sequences revealed a discrete distribution in sperm nuclei. Histones localized to the posterior ring region （i.e. the sperm nuclear annulus）, whereas PRM1 and PRM2 appeared to be dispersed throughout the entire nucleus. Conclusion： The co-localization of the human core sperm histones with the telomeric regions of chromosome 16 is consistent with the reorganization of specific non-protamine regions into a less compacted state.

Comparative study of the effects of three semen preparation media on semen analysis,DNA damage and protamine deficiency,and the correlation between DNA integrity and sperm parameters

Semen samples collected from 28 male partners of infertile couples were divided into three equal aliquots and prepared with three selected media,such as PureSperm （Nidacon,Gothenburg,Sweden）,Sil-Select Plus^TM （Fertipro,Beemem,Belgium） and SpermGrad^TM（Vitrolife,Gothenburg,Sweden）. The differences in mean percentages of semen parameters were assessed by repeated measures analysis. Correlations of sperm DNA damage,as measured by terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay,and of protamine deficiency,as measured by chromomycin A3 （CMA3） staining with sperm parameters,were determined by Pearson＇s correlation. After preparation with all three media,sperm concentrations decreased （P〈0.05） while percentages of sperm with normal morphology increased （P〈0.05）. Percentages of sperm motility,rapid motility and progressive motile concentration （PMC） increased （P〈0.05） for each ofthese parameters,PureSperm preparation gave the best results （P〈0.05）. The percentage of DNA damage decreased in the PureSperm and Sil-Select Plus preparations （17.9% and 31.3%,respectively,P〈0.05） and increased in the SpermGrad preparation （56.3%,P〈0.05）. Protamine deficiency also decreased in all three kinds of media,59.3%,47.7% and 40.3% for PureSperm,Sil-Select Plus and SpermGrad preparations,respectively （P〈0.05）. The percentage of DNA-damaged sperm was negatively correlated with the percentages of sperm motility,rapid motility and PMC,but was positively correlated with static motility （P〈0.05）. This comparative study and correlation analysis revealed that PureSperm preparation yielded sperm with the best motility and the lowest percentage of protamine deficiency. The Sil-Select Plus preparation yielded sperm with the lowest amount of DNA damage. The SpermGrad preparation had a high percentage of sperm with normal morphology,but also had the highest percentage of sperm with DNA damage. Sperm DNA damage was correlated with percentages of sperm motility,rapid motility,static motility and PMC.

Human protamines and the developing spermatid: their structure, function, expression and relationshipwith male infertility

<abstract>During spermiogenesis, the protamine proteins play an integral role in spermatid chromatin compaction. Recent research has focused on many facets of protamine biology, including protamine gene and protein structure/ function relationships, mechanisms of protamine expression regulation and involvement of the protamines in male fertility. In this paper, we review our current understanding of the structure and function of the protamine-1 (P1) and protamine-2 (P2) proteins and genes, the expression and regulation of these genes and the relationship between the protamines and male fertility. In addition, we offer a brief outlook on future investigation into protamine proteins.

The Protamine-like DNA-binding Protein P6.9 Epigenetically Up-regulates Autographa californica Multiple Nucleopolyhedrovirus Gene Transcription in the Late Infection Phase

Protamines are a group of highly basic proteins first discovered in spermatozoon that allow for denser packaging of DNA than histones and will result in down-regulation of gene transcription^l~. It is well recognized that the Autographa californica multicapsid nucleopolyhedrovirus （AcMNPV） encodes P6.9, a protamine-like protein that forms the viral subnucleosome through binding to the viral genome[29]. Previous research demonstrates that P6.9 is essential for viral nucleocapsid assembly, while it has no influence on viral genome replication1311. In the present study, the role of P6.9 in viral gene transcription regulation is characterized. In contrast to protamines or other protamine-like proteins that usually down-regulate gene transcription, P6.9 appears to up-regulate viral gene transcription at 12-24 hours post infection （hpi）, whereas it is non-essential for the basal level of viral gene transcription. Fluorescence microscopy reveals the P6.9＇s co-localization with DNA is temporally and spatially synchronized with P6.9＇s impact on viral gene transcription, indicating the P6.9-DNA association contributes to transcription regulation. Chromatin fractionation assay further reveals an unexpected co-existence of P6.9 and host RNA polymerase II in the same transcriptionally active chromatin fraction at 24 hpi, which may probably contribute to viral gene transcription up-regulation in the late infection phase.

Basic peptide protamine exerts antimicrobial activity against periodontopathic bacteria——Growth inhibition of periodontopathic bacteria by protamine

Protamine was investigated for its antibacterial activity against the periodontal pathogens, Porphyromonas gingivalis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans. We determined the minimum inhibitory concentrations of protamine and its hydrolysate and their bactericidal activity. Protamine inhibited the growth of all periodontopathic bacteria tested on agar plates. Protamine, which MIC was 6.3 × 10-7 g L-1, was most effective against P. gingivalis. The antibacterial effect of native protamine was higher than that of its hydrolysate. An ATP bioluminescence assay revealed that protamine showed bactericidal activity against P. gingivalis in a time-dependent manner. These results indicate that protamine could be candidate peptide for prevention of P. gingivalis infection.

Studies on Expression of P1 Protamine Gene in Rat and Mouse Testis

Protamine is a kind small, basic protein rich in arginine residues and found to be complexed with DNA in spermatozoa. We have cloned a 150 bp cDNA encoding the rat protamine (rP) by RT PCR technique. Dig labelled cDNA for rP was used for Northern blot analysis to study the expression of P1 protamine gene in rat and mouse. P1 protamine mRNA was detected only in rat testis, no hybridization signals were detected in rat brain and lever. In addition, the presence of P1 protamine mRNA was detected not only in rat testis, but also in mouse testis. Dig labelled cDNA for mouse protamine 1 (mP1) was used to study the expression of mP1 gene during the process of sexual maturation of mouse. 7～8 d after birth, no mP1 mRNA could be detected. At d 24～26, mP1 mRNA was detectable migrating as a homogeneous band at 580 nucleotides, whereas in sexually mature animals, a heterogeneous mixture of RNAs ranging from 450～580 bases in length was observed. Histological studies revealed that in the testis of 7～8 day old mouse, spermatogenesis has developed to the spermatocyte stage, whereas round spermatids (Rs) were present in the testis of the mice with 24～26 d age and elongating spermatids (Es) were present in the testis of sexually mature animals. Electrophoresis of total nuclear basic proteins (TNBP) revealed that the Rs could possess the somatic histones, while Es was found to have protamine and less histone. These results indicate that the P1 protamine gene is tissues specifically expressed and the P1 protamine is showing to be conservative in evolution. During the process of sexual maturation, along with morphological changes, mP1 gene was transcribed in Rs and translated in Es. The mechanism of protamine gene expression was discussed.

Studies on P_1, P_2 Protamine mRNA in Sperms of Human,Rat and Mouse

ve To investigate the existence of the protamine mRNA in sperms of human, rat and mouse

Influence of N-acetylcysteine on pituitary-gonadal axis hormones and protamine expression level in streptozotocin-induced diabetic male rats

Objective:To study the influence of N-acetylcysteine on the pituitary-gonadal axis hormones and protamine expression level in streptozotocin-induced diabetic male rats.Methods:Forty-two adult male Wistar rats were divided into 6 groups,with 7 rats in each group.The control group left untreated;the streptozotocin group only received 50 mg/kg body weight streptozotocin intraperitoneally for 5 days to induce diabetes;the N-acetylcysteine group only received 200 mg/kg body weight N-acetylcysteine intraperitoneally,and the streptozotocin+N-acetylcysteine groups 1,2 and 3 received 50 mg/kg streptozotocin intraperitoneally for 5 days to induce diabetes and then received 100,200 and 400 mg/kg body weight doses of N-acetylcysteine intraperitoneally for 28 days,respectively.Enzyme-linked immunosorbent assay was used to measure the serum levels of luteinizing hormone(LH),follicle-stimulating hormone(FSH)and testosterone,and real-time PCR was applied for measuring protamine expression level.Results:Compared to the control and N-acetylcysteine groups,a significant decrease in the body weight,testicular weight and levels of testosterone and protamine expression was observed in the streptozotocin group and the streptozotocin+N-acetylcysteine groups 1 and 2.On the contrary,the levels of LH and FSH increased significantly.In the streptozotocin+N-acetylcysteine group 3,the body weight,testicular weight and expression level of protamine were significantly higher than those of the streptozotocin group.In the streptozotocin+N-acetylcysteine groups,testosterone and LH levels were significantly higher than and lower than the streptozotocin group,respectively.In the streptozotocin+N-acetylcysteine groups 2 and 3,the level of FSH was significantly lower than that of the streptozotocin group and streptozotocin+N-acetylcysteine group 1.Furthermore,a significant increase in the expression level of protamine was observed in the streptozotocin+N-acetylcysteine groups 2 and 3 when compared to the streptozotocin group and streptozotocin+N-acetylcysteine group 1.Conclusions:N-acetylcysteine in an optimal dose of 400 mg/kg body weight has a protective influence on the pituitary-gonadal axis hormones and also on the expression level of protamine in diabetic male rats.

Neutral protamine hagedorn/regular insulin in the treatment of inpatient hyperglycemia: Comparison of 3 basal-bolus regimens

AIM To compare the safety and efficacy or 3 basal-bolus regimens of neutral protamine hagedorn(NPH)/regular insulin in the management of inpatient hyperglycemia.METHODS We randomized 105 patients with blood glucose levelsbetween 140 and 400 mg/dL to a basal-bolus regimen of NPH insulin given once(n = 30), twice(n = 40) or three times(n = 35) daily, in addition to pre-meal regular insulin. Major outcomes included were differences in glycemic control, frequency of hypoglycemia and total insulin dose.RESULTS NPH insulin given in a once-daily regimen was associated with better glycemic control(58.3%) compared to twice daily(42.4%) and three times daily(48.9) regimens(P = 0.031). The frequency of hypoglycemia was similar between the three groups(2.0%, 0.7% and 1.2%, P = 0.21). The mean insulin dose at discharge was 0.48 ± 0.14 U/kg in the once-daily group compared to 0.69 ± 0.28 in the twice-daily, and 0.65 ± 0.20 in the three times daily regimens(P < 0.001).CONCLUSION NPH insulin administered in a once-daily regimen resulted in improvement in glycemic control with similar rates of hypoglycemia compared to a twice-daily and a three times-daily regimen. Further studies are needed to evaluate whether this regimen could be implemented in all hospitalized patients with hyperglycemia.

Aberrant protamination in sperm correlates to anomalous nuclear and cytoplasmic architectures in infertile males with sperm dysmorphology

Aberrant sperm protamination is linked to sperm dysmorphology and nuclear chromatin condensation.Yet,its effects on sperm cytoplasmic maturation remain largely unexplored.The relationships of protamines,sperm morphology,DNA damage,and cytoplasmic remodeling were illustrated in this study to provide fresh perspectives on the mechanisms of male infertility.A total of 205 infertile males were allocated into 5 groups according to the percentage of spermatozoa exhibiting abnormal morphology within their samples.Sperm concentration,motility,abnormal sperm morphology,cytoplasmic droplets(CDs),and excess residual cytoplasm(ERC)were analyzed according to the World Health Organization manual(2010).Sperm nuclear vacuoles(NVs)were determined by propidium iodide(PI)staining.Sperm protamine expressions(P1 and P2)were detected by western blot.DNA damage was measured by acridine orange test(AOT)to calculate the proportion of sperm with single-strand DNA breaks(SSBs).Our data showed that sperm concentration and motility in infertile males significantly decreased with the severity of abnormal sperm morphology(both P<0.01).P1 level,P1/P2 ratio,and SSB rate increased with the severity of sperm dysmorphology,whilst the P2 level decreased(all P<O.01).NVs,CDs,and ERC were more common in males with sperm dysmorphology and positively correlated with the SSB rate(all P<O.01).The relationships between the SSB rate and the P1/P2 ratio were also significant(P<0.01).Aberrant protamination may cause sperm dysmorphology and compromise male fertility by impairing sperm's nucleus and cytoplasm maturation,with the P1/P2 ratio potentially serving as a valuable indicator of sperm quality and male fertility.

Comparison of ART outcomes in men with altered mRNA protamine 1/protamine 2 ratio undergoing intracytoplasmic sperm injection with ejaculated and testicular spermatozoa

Assisted reproductive technologies invoIving the use of spermatozoa and eggs for in vitro fertilization(IVF)have come as the solution for many infertile couples to become parents.However,in some cases,the use of ejaculated spermatozoa delivers poor IVF performance.Some studies have suggested the use of testicular spermatozoa in severe male in fertility cases,but no guideli nes regarding their utilization are currently available.In the present study,we found the mRNA protamine 1/protamine 2(P1/P2)ratio to be a valuable biomarker of poor sperm function that could be used as a diagnostic key for the identification of cases that would benefit from the use of testicular spermatozoa.A total of 23 couples undergoing egg donation cycles with at least one previous cycle failure were studied.All couples underwent two consecutive intracytoplasmic sperm injection(ICSI)cycles with either ejaculated or testicular spermatozoa(TESA).The sperm mRNA P1/P2 ratio,fertilization rate,blastocyst rate,and pregnancy and live birth rate were compared.Results showed improved ICSI and clinical outcomes in cycles with testicular spermatozoa in men with altered mRNA P1/P2 ratios.TESA cycles presented significantly higher rates of fertilization(mean±standard deviation:76.1%±15.1%vs 65.5%±18.8%),blastocyst formation(55.0%±20.3%vs 30.8%±23.8%),and good morphological quality blastocyst(28.9%±22.9%vs 13.5%±17.9%)and also improvements on pregnancy(60.9%vs 0%)and healthy birth rates(56.5%vs 0%)than EJACULATE cycles.The results described here suggest that in patients with previous IVF/ICSI failures and aberrant mRNA protamine ratios,the use of testicular spermatozoa may be a good alternative to improve clinical outcomes.

Chromatin condensation but not DNA integrity of pig sperm is greater in the sperm-rich fraction

Background Protamination and condensation of sperm chromatin as well as DNA integrity play an essential role during fertilization and embryo development.In some mammals,like pigs,ejaculates are emitted in three separate fractions:pre-sperm,sperm-rich(SRF)and post sperm-rich(PSRF).These fractions are known to vary in volume,sperm concentration and quality,as well as in the origin and composition of seminal plasma(SP),with differences being also observed within the SRF one.Yet,whether disparities in the DNA integrity and chromatin condensation and pro-tamination of their sperm exist has not been interrogated.Results This study determined chromatin protamination(Chromomycin A3 test,CMA_(3)),condensation(Dibromobi-mane test,DBB),and DNA integrity(Comet assay)in the pig sperm contained in the first 10 m L of the SRF(SRF-P1),the remaining portion of the sperm-rich fraction(SRF-P2),and the post sperm-rich fraction(PSRF).While chromatin protamination was found to be similar between the different ejaculate fractions(P>0.05),chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF(P=0.018 and P=0.004,respectively).Regarding DNA integrity,no differences between fractions were observed(P>0.05).As the SRF-P1 has the highest sperm concentra-tion and ejaculate fractions are known to differ in antioxidant composition,the oxidative stress index(OSi)in SP,calcu-lated as total oxidant activity divided by total antioxidant capacity,was tested and confirmed to be higher in the SRF-P1 than in SRF-P2 and PSRF(0.42±0.06 vs.0.23±0.09 and 0.08±0.00,respectively;P<0.01);this index,in addition,was observed to be correlated to the sperm concentration of each fraction(Rs=0.973;P<0.001).Conclusion While sperm DNA integrity was not found to differ between ejaculate fractions,SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the PSRF.This could be related to the OSi of each fraction.

精细胞中促进组蛋白-鱼精蛋白替换的分子机制研究进展

在精子形成过程中,圆形精子细胞核内染色质中的组蛋白逐渐被鱼精蛋白替代,使染色质凝聚得更为紧凑,奠定成熟精子头部特异形态的结构基础。若组蛋白-鱼精蛋白替换缺陷,将导致精子核凝聚失败,表现出精子形态异常和活力降低,受精困难。研究显示,组蛋白翻译后修饰、瞬时DNA链断裂、DNA链断裂修复和染色质重塑复合体识别等一系列变化都会促进组蛋白-鱼精蛋白的替换,本文从以上几个方面综述了目前对于促进组蛋白-鱼精蛋白替换分子机制的研究进展,以期为雄性不育症的诊断和治疗提供理论基础。

信阳毛尖多酚-鱼精蛋白复合保鲜剂对冷藏鳜鱼鱼丸品质的影响

为提高鳜鱼鱼丸在低温贮藏时的品质,在鱼丸制作过程中分别加入不同组成成分的天然保鲜剂(0.2%的信阳毛尖多酚、0.3%鱼精蛋白和二者复合物),制成的鱼丸置于4℃下贮藏。定期测定贮藏期间不同保鲜剂处理的鱼丸菌落总数、TVB-N值、TBA值、甲醛含量、三甲胺含量、白度、凝胶强度及感官评分,以评价不同保鲜剂对鱼丸在贮藏期间的品质改善效果。实验结果显示:信阳毛尖多酚和鱼精蛋白均具有遏制微生物生长、减缓蛋白质降解和脂肪氧化速率、降低贮藏过程中甲醛和三甲胺生产量、维持鱼丸蛋白凝胶强度的功能。而两种保鲜剂复配剂对鱼丸的保鲜效果最好,极大地提升了贮藏期间鱼丸的品质并延长了货架期。

精蛋白重组人胰岛素混合注射液40/60治疗老年2型糖尿病的疗效及对患者预后的影响

目的:探讨精蛋白重组人胰岛素混合注射液40/60(简称甘舒霖40/60)治疗老年2型糖尿病(T2DM)的疗效及对患者预后的影响。方法:采用简单随机分组法将西部战区总医院2022年12月至2023年12月收治的106例T2DM患者分为对照组(n=53)和治疗组(n=53)。对照组予常规二联降糖方案治疗(二甲双胍+达格列净),治疗组予甘舒霖40/60皮下注射治疗。12周后评价疗效及安全性,测定两组治疗前、后血糖指标、体重指数(BMI)、胰岛β细胞功能指标及血清单核细胞趋化因子(MCP-1)、超敏C反应蛋白(hs-CRP)、白细胞介素6(IL-6)水平差异,并统计随访6个月预后。结果:治疗组治疗总有效率为90.57%,较对照组的71.70%更高(P<0.05)。治疗后,与对照组相比,治疗组空腹血糖(FPG)、餐后2h血糖(2hPG)、糖化血红蛋白(HbAlc)、胰岛素抵抗指数(HOMA-IR)及血清MCP-1、hs-CRP、IL-6水平更低,胰岛β细胞功能指数(HOMA-β)更高(P<0.05)。治疗组(3.77%)、对照组(7.55%)总不良反应发生率比较差异无统计学意义(P>0.05);两组随访6个月微血管并发症发生率差异无统计学意义(P>0.05)。结论:采用甘舒霖40/60皮下注射治疗老年T2DM安全有效,不仅能降低患者血糖水平,促进胰岛β细胞功能恢复,减轻炎症反应,但并未改善患者短期预后。

地特胰岛素与精蛋白人胰岛素治疗妊娠期糖尿病的疗效及对患者血糖水平的影响

目的分析地特胰岛素与精蛋白人胰岛素治疗妊娠期糖尿病的疗效及对患者血糖水平的影响。方法回顾性选取2023年2月—2024年3月泉州市第一医院收治的110例妊娠期糖尿病患者的临床资料,根据治疗方式的不同分为两组,各55例。对照组皮下注射精蛋白人胰岛素,研究组皮下注射地特胰岛素,对比两组临床疗效、血糖水平、胰岛功能及不良反应发生率。结果研究组治疗总有效率高于对照组,差异有统计学意义(P<0.05).治疗后,研究组糖化血红蛋白、空腹血糖、餐后2 h血糖水平均低于对照组,差异有统计学意义(P均<0.05)。治疗后,研究组胰岛β功能指数高于对照组,胰岛素抵抗指数低于对照组,差异有统计学意义(P均<0.05)。研究组不良反应发生率低于对照组,差异有统计学意义(P<0.05)。结论地特胰岛素在治疗妊娠期糖尿病方面具有长效平稳降糖效果,能够改善胰岛功能及降低不良反应发生风险。

国内外精蛋白重组人胰岛素注射液的有关物质比较

目的精蛋白重组人胰岛素适用于2型糖尿病患者的治疗,现行标准中仅对本品有关物质A_(21)脱氨人胰岛素进行控制,未进行B_(3)和B_(3)iso脱氨人胰岛素限度的控制。采用两种方法对精蛋白重组人胰岛素注射液中的有关物质进行测定并比较国内外共17批样品的质量。方法采用高效液相色谱法梯度洗脱,色谱柱:Waters Xbridge C_(18)4.6 mm×250 mm(5μm),检测波长:214 nm,柱温:40℃,流速:1.0 ml/min,进样体积:20μl;《中国药典》2020年版方法:流动相A为0.2 mol/L硫酸钠溶液(pH值2.3)-乙腈(82∶18),流动相B为乙腈-水(50∶50);某企业自拟方法:流动相A为0.2 mol/L硫酸钠溶液(pH值3.6)-乙腈(90∶10),流动相B为乙腈-水(50∶50)。结果国内外样品的A_(21)脱氨人胰岛素含量差异不大、国内一家企业B_(3)和B_(3)iso脱氨人胰岛素含量低于其他企业,国内另一家企业有关物质总和高于其他企业;两种方法检出A_(21)脱氨人胰岛素的含量差异无统计学意义(P>0.05),企业自拟方法能检测出更多的有关物质。结论两种方法均快速、简便,可用于比较国内外样品中的有关物质,同时为企业在制定杂质控制限度时提供依据。

成人骨髓间质干细胞分化为神经元样细胞前后基因表达的研究

目的探讨成人骨髓间质干细胞(MSC)分化为神经元样细胞前后基因表达的变化。方法从成人骨髓分离MSC,培养扩增。用参芪液诱导hMSC分化为神经元,并用免疫组化法检测神经元烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)的表达来鉴定是否为神经元样细胞。采用半定量RTPCR方法检测10个基因(包括内胚层基因Ceruloplasmin;中胚层基因SM22;外胚层基因Amyloidprecursorprotein、syntaxin;生殖系基因protamine;神经元特异性基因NeuroD、NF、NSE、GFAP、Tau)在诱导分化前后的变化。结果hMSC经参芪液诱导后,可见神经元样细胞。免疫组化结果显示诱导出的神经元样细胞表达NSE、NF阳性,GFAP阴性。hMSC在分化前表达内胚层、中胚层、外胚层和生殖系基因,随着分化时间的延长,生殖系基因不表达,内胚层和中胚层基因表达减弱,外胚层基因表达基本不变。而在分化过程中外胚层基因和神经元特异性标记基因表达明显增强。结论hMSC分化为神经元样细胞的过程并不是简单的神经特异性基因的“开”和“关”,而是与神经细胞分化成熟过程中相关基因表达的量相关。