Osteocalcin fragment in bone matrix enhances osteoclast maturation at a late stage of osteoclast differentiation

Although 14-day-old mouse embryonic calvarial cells cultured in plastic culture dishes in the presence of 1,25-dihydroxyvitamin D₃[1,25-(OH)₂D₃] for 7 days could barely resorb bone slices, the same calvarial cells cultured with an ethylenediaminetetraacetic acid (EDTA) extract from bovine bone powder under the same conditions stimulated pit formation on bone slices in a dose-dependent manner. Therefore, the present study was conducted to purify and characterize this osteoclast maturation-inducing factor(s) from the bone matrix. The protein having osteoclast maturation-inducing activity in the EDTA extract was purified by gel filtration over Superdex 75 preparation grade and chromatography on hydroxyapatite, Mono Q, and C8 reversed-phase HPLC by monitoring the ability of the eluted fractions to elicit pit formation on bone slices. The molecular weight of the purified protein estimated by high-resolution polyacrylamide gel electrophoresis was 5.7kDa and 6.8kDa in the respective absence and presence of 2-mercaptoethanol. The sequence of the 30-amino-acid purified protein corresponded to the 7th to 36th residues of bovine osteocalcin. The osteocalcin fragment, missing the initial 6 residues at the N-terminal region, exhibited higher osteoclast maturation-inducing ability than bovine intact osteocalcin on a per weight basis. The osteocalcin fragment had no effect on the expression of receptor activator of nuclear factor (NF)-B ligand (RANKL) and osteoprotegerin (OPG) genes in calvarial cells, nor did it enhance the bone-resorbing activity of mature osteoclasts. When the osteocalcin fragment was added to late-stage (days 4–7) or to early-stage (days 0–3) cultures of calvarial cells pretreated with 1,25-(OH)₂D₃, its stimulatory effect was observed in the late-stage cultures rather than in the early-stage ones. In addition, the osteocalcin fragment directly enhanced the formation of osteoclasts with bone-resorbing ability from Mac-1⁺ c-Fms⁺ cells in the presence of macrophage colony-stimulating factor (MCSF) and RANKL. These results suggest that the osteocalcin fragment in bone matrix is involved in osteoclast maturation, especially at a late stage of osteoclast differentiation.     

下颌后缩与前突青少年患者牙龄差异性分析

目的:通过对下颌后缩与下颌前突两类青少年患者牙齿发育进程的差异性分析,探讨生长发育期儿童牙齿发育早晚对下颌矢状向骨型的影响.方法:随机选择错(牙合)患者143(男80例,女63)例,年龄9～12.9(平均11.5)岁,依据头颅侧位片上SNB角、SNA角以及ANB角的测量结果,分为下颌后缩组(SNA正常,SNB＜77.6,ANB≥5)与下颌前突组(SNA正常,ANB＜0).在全口曲面断层片上应用Demirjian评价牙龄方法评定样本牙齿成熟度并转化为牙龄,比较两组间牙龄的差异性.结果:下颌后缩组患者的平均牙龄为11.9岁,牙龄年龄差为0.3岁;下颌前突组患者的平均牙龄为11.5岁,牙龄年龄差为0.1岁.两组牙龄之间未发现明显的差异性;下颌后缩患者的牙齿发育相对于下颌前突患者有轻微提前的趋势.结论:下颌后缩与下颌前突两类青少年患者的牙龄没有显著性差异,提示牙齿发育的早晚对下颌矢状向骨型不存在明显的相关性.     

度他雄胺对大鼠附睾精子成熟和生育的影响

目的通过度他雄胺对大鼠附睾精子和生育的影响,探索调节雄性生育的睾丸后作用靶点。方法使用度他雄胺20和40 mg/(kg.d)大鼠灌胃给药,连续2周。给药结束后雄雌鼠按1∶2合笼,计算生殖指数;采用计算机辅助精子分析系统分析精子活力和形态;采用SYBR-14和PI双重荧光染色计算精子存活率;采用Elisa法测定大鼠睾酮(T)和双氢睾酮(DHT)血清浓度;采用HE染色法对各组睾丸、附睾进行组织学分析。结果度他雄胺低、高剂量组双氢睾酮浓度均显著下降,分别为0.54和0.28 nmol/L(P<0.01),精子活力明显降低,分别为39.0%和28.7%(P<0.01),畸形率分别增加为10.3%和15.6%(P<0.05),最后受孕率分别降为62.5%和38.4%。而睾酮水平和交配指数均无明显变化(P>0.05),睾丸和附睾亦无明显病理学改变。结论度他雄胺通过抑制DHT生成,影响附睾精子成熟而导致大鼠不育,为今后男性避孕和不育药物研发提供了新思路。     

体外受精-胚胎移植足月妊娠胎盘中表皮生长因子受体的表达

①目的探讨体外受精-胚胎移植(IVF-ET)足月妊娠胎盘中表皮生长因子受体(EGFR)的表达及其变化。②方法取IVF-ET足月分娩的胎盘组织标本30例,并以30例相同孕周自然妊娠分娩的胎盘组织标本为对照组,应用免疫组织化学SP法染色,通过Fromowitz综合计分法检测EGFR。③结果EGFR主要表达于胎盘的合体滋养细胞(ST)的胞膜和胞浆中,染色强度以刷状缘最强,IVF-ET组胎盘EGFR染色强度较正常对照组增强,差异有统计学意义(P<0.05);IVF-ET组胎盘重量和新生儿体重与正常对照组比较,无显著性差异(P>0.05);新生儿出生体重与胎盘重量呈高度相关性(r=0.709,P<0.01)。④结论IVF-ET组胎盘EGFR表达增强,可能与COH药物作用或黄体支持中母体体内超生理剂量孕酮的作用有关。     

In-vitro Maturation of Immature Oocytes from Preantral Follicles in Prepuberal Mice

Objective To observe the morphological changes in in vitro growth of preantral follicle isolated from prepuberal mice and to assess impacts of gonadotropin （Gn）, insulin transferrin selenium （ITS） and epidermal growth factor （EGF） on their development. Methods Early preantral mice follicles （90-130μm diameter） were mechanical isolated and selected from 2 weeks＇ old mice and then cultured in alpha-minimal essential medium （α-MEM） with or without Gn, ITS and EGF. The preantral follicles were cultured singly in 20 miovliters droplets for up to 14 d. The medium was replaced and the .follicles were observed everyday. Granulosa cells （GC） prolification, antrum formation and oocyte maturation were recorded. Results The medium with Gn supported preantral follicle culture in vitro, during which they retained a three-dimensional structure, maintained oocytes viability and increased in diameter and number of somatic cells＇. Preantral follicles cultured in Gn medium grew obviously, while those without Gn grew slowly and after 6 d＇s culture began to shrink and blacken. Significant increase in survival rate and maturation rate of oocytes was observed in Gn group （P〈0.01）, with 92.9% survived and 28.7%formed an antrum. Further supplementation of the Gn medium with ITS and rLH, resulted in the significant increase in survival and maturation of preantral follicle （P〈0.05） Conclusions α-MEM can be the medium for in vitro culture （IVC） of preantral follicles, but need to be added with rLH/rFSH, rHCG/rEGF to facilitate thecal cell attachment, GC proliferation and oocyte maturation.     

颈椎骨量化法判断天津地区青少年生长发育状况

【目的】应用量化方法对天津地区10～15岁青少年颈椎骨发育状况进行研究分析，寻找其变化趋势，确定标准值范围，帮助临床医生更准确地判断个体骨骼的成熟度。【方法】选取10～15岁患者238例，其中男113例，女125例，拍摄头颅定位侧位片，选用WinCeph8．0头影测量分析与图像数据管理系统测量第三、四颈椎体中部的高度和前后径长度，求其比值H3／AB和H4／AP4，并进行统计学分析。【结果】10—15岁儿童在生长发育阶段其颈椎H3／AB、H4／AP4值的均数呈明显的上升趋势，且无性别差异，得出不同阶段的均值范围及其变化曲线图。【结论】现阶段天津地区青少年颈椎H3／A岛、H4／AP4值变化趋势与青春生长发育期具有紧密的对应关系，在临床中可以用颈椎H3／AR、H4／AP4均值范围及其变化曲线图，对青春生长发育期所在阶段准确量化的预测和评价。     

BCMA靶向的嵌合抗原受体T细胞治疗复发/难治多发性骨髓瘤患者发生肿瘤溶解综合征的危险因素

目的：探究应用B细胞成熟抗原（BCMA）靶向的嵌合抗原受体（CAR）T细胞治疗的复发/难治多发性骨髓瘤（MM）患者发生肿瘤溶解综合征（TLS）的危险因素。方法：收集浙江大学医学院附属第一医院2018年7月至2021年12月共99例接受BCMA靶向的CAR-T细胞治疗MM患者的临床资料,通过单因素分析及多因素logistic回归分析患者接受BCMA靶向的CAR-T细胞治疗后发生TLS的危险因素。结果：99例患者中,17例发生TLS（TLS组）,发生率为17.2%,发生时间为BCMA靶向的CAR-T细胞输注后（8.9±3.0）d。TLS组均出现TLS相关临床表现,其中出现肾功能不全17例,心律失常8例。TLS组均发生细胞因子释放综合征（CRS）,发生时间为BCMA靶向CAR-T细胞输注后1.0（1.0,6.5）d,其中3~4级CRS 13例。TLS组治疗前血肌酐、血尿酸较非TLS组高,3~4级CRS患者的比例也高于非TLS组（P<0.01或P<0.05）。Logistic回归分析结果显示,高血肌酐水平（OR=1.015,P<0.01）和严重CRS（OR=9.371,P<0.01）是TLS发生的独立危险因素。结论：接受BCMA靶向的CAR-T细胞治疗的复发/难治MM患者具有较高的TLS发生率,高血肌酐水平和严重CRS是TLS的主要危险因素,临床可通过降低血肌酐、控制CRS严重程度预防TLS的发生。     

Influence of epididymal maturation on cyclic AMP levels in hamster spermatozoa

Whilst in their native epididymal fluid, sperm from the caput epididymis of the rat and hamster contain significantly (P less than 0.01) greater amounts of cAMP than do sperm from the cauda epididymis. The cAMP levels in both cell types from these species underwent a rapid increase concomitant with dilution to a density of 20 x 10(6)/ml. Further analyses in the hamster indicated that this increase was calcium-dependent, and could be enhanced by treatment with the calmodulin antagonist, calmidazolium. Dilution of hamster sperm to a concentration of 1 x 10(6)/ml was not associated with a rapid rise in cAMP levels. This effect was shown to be due to the dilution of a component in epididymal plasma. When incubated at this lower density, the cAMP content of hamster caput sperm remained low over a 3 h period, whilst similarly treated caudal sperm exhibited a progressive rise in cAMP levels. Thus, in contrast to other species, cAMP does not appear to play a pivotal role in acquisition of the capacity for movement during epididymal maturation in the rat and hamster. However, this nucleotide may be involved in the post-ejaculatory modifications of motility which accompany the terminal stages of capacitation.     

Blimp1-BCMA调控浆细胞功能在系统性红斑狼疮发病中的意义

【摘要】 目的 探讨Blimp1在MRL/lpr狼疮鼠中的表达水平,为靶向浆细胞治疗SLE提供思路。方法用EMSA和CHIP证实Blimp1对B细胞成熟抗原（BCMA）的调控作用,并用实时定量PCR检测MRL/lpr狼疮鼠与正常鼠脾脏和淋巴结中Blimp1mRNA的表达差异。结果 Blimp1能与BCMA基因启动子相结合。狼疮鼠组脾脏Blimp1mRNA的相对表达水平高于正常对照组Blimp1mRNA的相对表达水平（Z = 2.609,P < 0.01）,狼疮鼠组淋巴结中Blimp1的相对表达水平也高于正常对照组Blimp1mRNA的相对表达水平（ Z = 3.402,P < 0.01）,差异有统计学意义。结论 BCMA可能是Blimp1的靶基因,Blimp1可直接调控BCMA表达,在维持浆细胞存活及抗体分泌中发挥作用。 【关键词】 红斑狼疮,系统性; Blimp1; B细胞成熟抗原; 浆细胞; 小鼠,近交MRL lpr