Satellite Cells Exhibit Decreased Numbers and Impaired Functions on Single Myofibers Isolated from Vitamin B6-Deficient Mice

Emerging research in human studies suggests an association among vitamin B6, sarcopenia, and muscle strength. However, very little is known regarding its potential role at the cellular level, especially in muscle satellite cells. Therefore, to determine whether vitamin B6 affects the satellite cells, we isolated single myofibers from muscles of vitamin B6-deficient and vitamin B6-supplemented mice. Subsequently, we subjected them to single myofiber culture and observed the number and function of the satellite cells, which remained in their niche on the myofibers. Prior to culture, the vitamin B6-deficient myofibers exhibited a significantly lower number of quiescent satellite cells, as compared to that in the vitamin B6-supplemented myofibers, thereby suggesting that vitamin B6 deficiency induces a decline in the quiescent satellite cell pool in mouse muscles. After 48 and 72 h of culture, the number of proliferating satellite cells per cluster was similar between the vitamin B6-deficient and -supplemented myofibers, but their numbers decreased significantly after culturing the myofibers in vitamin B6-free medium. After 72 h of culture, the number of self-renewing satellite cells per cluster was significantly lower in the vitamin B6-deficient myofibers, and the vitamin B6-free medium further decreased this number. In conclusion, vitamin B6 deficiency appears to reduce the number of quiescent satellite cells and suppress the proliferation and self-renewal of satellite cells during myogenesis.     

Is mandible derived mesenchymal stromal cells superior in proliferation and regeneration to long bone-derived mesenchymal stromal cells?

Mesenchymal stromal cells (MSCs) are cells with the characteristic ability of self-renewal along with the ability to exhibit multilineage differentiation. Bone marrow (BM) is the first tissue in which MSCs were identified and BM-MSCs are most commonly used among various MSCs in clinical settings. MSCs can stimulate and promote osseous regeneration. Due to the difference in the development of long bones and craniofacial bones, the mandibular-derived MSCs (M-MSCs) have distinct differentiation characteristics as compared to that of long bones. Both mandibular and long bone-derived MSCs are positive for MSC-associated markers such as CD-73, -105, and -106, stage-specific embryonic antigen 4 and Octamer-4, and negative for hematopoietic markers such as CD-14, -34, and -45. As the M-MSCs are derived from neural crest cells, they have embryogenic cells which promote bone repair and high osteogenic potential. In vitro and in vivo animal-based studies demonstrate a higher rate of proliferation and high osteogenic potential for M-MSCs as compared to long-bones MSCs, but in vivo studies in human subjects are lacking. The BM-MSCs have their advantages and limitations. M-MSCs may be utilized as an alternative source of MSCs which can be utilized for tissue engineering and promoting the regeneration of bone. M-MSCs may have potential advantages in the repair of craniofacial or orofacial defects. Considering the utility of M-MSCs in the field of orthopaedics, we have discussed various unresolved questions, which need to be explored for their better utility in clinical practice.     

Assessment of Estrogen Receptors and Apoptotic Factors in Cryopreserved Human Ovarian Cortex

The aims of this study were: (i) to examine frozen-thawed ovarian tissues for features of follicular health and atresia by histology; (ii) to assess the expression of estrogen receptors alpha (ERα) and beta (ERβ) by real-time PCR; (iii) to evaluate the Bax/Bcl-2 ratio, as an apoptotic index, in the ovarian tissues before and after cryopreservation.

Ovarian cortical biopsies were obtained from 11 patients. The fragments were subdivided into two groups, fresh (control tissues) and cryopreserved tissues obtained by direct plunging into liquid nitrogen. Both tissue groups were subjected to a histological evaluation of the healthy and atretic follicles, immunohistochemical localization of the ER, and a real-time PCR (qPCR) to evaluate the expression of ER, Bax, Bcl-2 as well as β-actin, as control gene. Damage was observed in 31% of primordial, 45% of primary, and 75% of secondary follicles in the cryopreserved tissue group. The qPCR analysis showed that the level of ERβ was greater in fresh than cryopreserved tissues, whereas the ERα expression and Bax/Bcl-2 ratio were similar in both tissue groups. A significant inverse association was observed between ERα mRNA levels in the fresh tissue group and subjects' ages.

The results show that cryopreservation and thawing of human ovarian tissue does not affect the morphology of primordial or primary follicles and that cryopreservation does not affect apoptosis. However, cryopreservation seems to have an inhibitory effect on the level of ERβ. Additional studies are needed to evaluate the differential effects of freezing follicles at different stages of follicular development and ovarian steroidogenesis.     

Bisphenol A diglycidyl ether induces adipogenic differentiation of multipotent stromal stem cells through a peroxisome proliferator-activated receptor gamma-independent mechanism

Background: Bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE), used in manufacturing coatings and resins, leach from packaging materials into food. Numerous studies suggested that BPA and BADGE may have adverse effects on human health, including the possibility that exposure to such chemicals can be superimposed on traditional risk factors to initiate or exacerbate the development of obesity. BPA is a suspected obesogen, whereas BADGE, described as a peroxisome proliferator–activated receptor gamma (PPARγ) antagonist, could reduce weight gain.Objectives: We sought to test the adipogenic effects of BADGE in a biologically relevant cell culture model.Methods: We used multipotent mesenchymal stromal stem cells (MSCs) to study the adipogenic capacity of BADGE and BPA and evaluated their effects on adipogenesis, osteogenesis, gene expression, and nuclear receptor activation.Discussion: BADGE induced adipogenesis in human and mouse MSCs, as well as in mouse 3T3-L1 preadipocytes. In contrast, BPA failed to promote adipogenesis in MSCs, but induced adipogenesis in 3T3-L1 cells. BADGE exposure elicited an adipogenic gene expression profile, and its ability to induce adipogenesis and the expression of adipogenic genes was not blocked by known PPARγ antagonists. Neither BADGE nor BPA activated or antagonized retinoid “X” receptor (RXR) or PPARγ in transient transfection assays.Conclusions: BADGE can induce adipogenic differentiation in both MSCs and in preadipocytes at low nanomolar concentrations comparable to those that have been observed in limited human biomonitoring. BADGE probably acts through a mechanism that is downstream of, or parallel to, PPARγ.     

Fertility of mice following receipt of ovaries slow cooled in dimethyl sulphoxide or ethylene glycol is largely independent of cryopreservation equilibration time and temperature

Cryopreservation procedures generally depend on both the cryoprotectant used and the equilibration conditions to which the material is exposed. The aim of the present study was to examine the effect of cryoprotectants (dimethyl sulphoxide (DMSO) and ethylene glycol (EG)) and equilibration conditions (0, 30 or 120 min at 0 degrees C or 120 min at room temperature) on the fertility of mice receiving cryopreserved mouse ovaries. The study compared the fertility of cryopreserved Day 14 mouse pup ovaries following grafting to adult recipient mice for 4 months. There was no effect of the cryoprotectant or equilibration condition used on the interval to the first plugging/mating or on the interval to the birth of the first litter, the size of litters, the number of litters produced or the total number of offspring produced. Despite this, when compared with control females (untreated, sham and fresh transplant) the cryopreservation and transplantation procedures delayed fertility. However, the size of litters was equivalent for all cryopreserved and control groups (P > 0.05). The results show that, for the equilibration conditions examined, DMSO and EG are equally efficient cryoprotective agents for mouse ovarian tissue.     

大鼠卵巢组织冷冻保存和自体移植后形态与功能的研究

目的:探讨大鼠卵巢组织冷冻、解冻和自体皮下移植后甾体激素分泌和卵泡生长发育的恢复情况。方法:将大鼠卵巢组织在程序性降温设备中缓慢冷冻,在液氮中保存14天后快速解冻。将新鲜和冻融后的大鼠卵巢组织分别自体异位移植于去势成年雌鼠的腹股沟皮下。术后每日行阴道细胞涂片检查,观察动情周期恢复情况,并分别于移植后4周和8周取移植卵巢进行组织学检查并测定血清雌孕激素水平。结果:新鲜组和冷冻组分别有80.6%和48.5%的大鼠恢复了动情周期,差异有统计学意义(P<0.01)。新鲜组和冷冻组首次恢复动情周期的天数分别为(8.72±1.69)天和(18.15±2.48)天,差异有统计学意义(P<0.01)。移植术后4周时冷冻组的E2和P水平与假手术组及新鲜组相比差异有统计学意义(P<0.05),而移植术后8周时3组激素水平差异已无统计学意义(P>0.05)。新鲜组和冷冻组移植术后4周和8周时移植物内均可见丰富的血管及不同发育阶段的卵泡和黄体。结论:大鼠的卵巢组织可以较好的耐受冷冻损伤,适宜条件下可以复苏恢复甾体激素的分泌,并且有卵泡的生长和发育。非血管吻合自体异位移植卵巢组织的方法简便易行且具有较高的成活率,是冻存卵巢组织较为安全有效的方法。     

Polyclonal antibodies to xenogeneic endothelial cells induce apoptosis and block support of tumor growth in mice

In this study, we demonstrate that vaccination of rabbits with murine endothelial cells yields polyclonal immunoglobulin (IgG) with potent antiangiogenic activity. The mechanism of this response appears to be through apoptosis of endothelial cells in vitro. Induction of polyclonal IgG in a xenogeneic host may be useful in passive immunotherapy of a variety of cancers. In fact, the antibody showed antitumor activity in three mouse tumor models (murine B16F10 melanoma, murine SVR angiosarcoma, and human DLD-1 colorectal adenocarcinoma). The polyclonal antibody generated here demonstrated utility in radioimaging of tumors in vivo, using positron emission tomography (PET) imaging, and suggested an antitumor effect in vivo. The results suggest that the antitumor effect in vivo may be related to antiangiogenic effects. Furthermore, anti-endothelial cell antibodies such as these could be useful reagents in isolating specific targets that comprise and induce the antiangiogenic effect.     

Would cancer stem cells affect the future investment in stem cell therapy

The common goal within the overwhelming interests in stem cell research is to safely translate the science to patients. Although there are various methods by which this goal can be reached, this editorial emphasizes the safety of mesenchymal stem cell (MSC) transplant and possible confounds by the growing information on cancer stem cells (CSCs). There are several ongoing clinical trials with MSCs and their interactions with CSCs need to be examined. The rapid knowledge on MSCs and CSCs has now collided with regards to the safe treatment of MSCs. The information discussed on MSCs can be extrapolated to other stem cells with similar phenotype and functions such as placenta stem cells. MSCs are attractive for cell therapy, mainly due to reduced ethical concerns, ease in expansion and reduced ability to be transformed. Also, MSCs can exert both immune suppressor and tissue regeneration simultaneously. It is expected that any clinical trial with MSCs will take precaution to ensure that the cells are not transformed. However, going forward, the different centers should be aware that MSCs might undergo oncogenic events, especially as undifferentiated cells or early differentiated cells. Another major concern for MSC therapy is their ability to promote tumor growth and perhaps, to protect CSCs by altered immune responses. These issues are discussed in light of a large number of undiagnosed cancers.     

间充质干细胞治疗肝病的机制

间充质干细胞(MSCs)的旁分泌机制是MSCs治疗肝病的重要机制.MSCs除了少量分化为肝细胞外,其分泌的一系列营养因子通过减轻炎症反应、抗肝细胞凋亡、抗纤维化、刺激内源性细胞分化增殖和新血管形成等发挥治疗肝病作用,但具体作用机制、有效治疗时间及潜在的副作用有待进一步阐明.此文对MSCs在减轻炎症反应、抗凋亡、抗纤维化等方面的进展进行了综述.     

Anti-major histocompatibility complex antibody responses in macaques via intradermal DNA immunizations

In simian immunodeficiency virus (SIV) models, immunization of macaques with uninfected human cells or human major histocompatibility complex (MHC) proteins can induce xenogeneic immune responses which can protect the animals from subsequent SIV challenges. These studies suggest that the induction of anti-MHC immune responses can be a viable vaccine strategy against human immunodeficiency virus type 1 (HIV-1). We have previously shown in mouse studies that DNA immunization with class I and class II MHC-encoding plasmids can elicit both xenogeneic and allogeneic antibody responses against conformationally intact MHC molecules (Vaccine 17 (1999) 2479-92). Here we take these observations one step closer to human applications and report that intradermal needle immunizations of non-human primates with plasmid DNA encoding human MHC alleles can safely elicit xenogeneic anti-MHC antibody responses. Moreover, injecting macaques with DNA encoding a specific macaque allogeneic MHC induced anti-allogeneic MHC antibodies production. These studies show that DNA immunization with MHC-encoding vectors can indeed be used to induce specific anti-human xenogeneic, as well as anti-macaque allogeneic MHC immunity in non-human primates. This strategy could thus be used to mobilize anti-MHC antibody response which may be useful as part of an anti-HIV-1 vaccination approach.     

Early postnatal food intake alters myofiber maturation in pig skeletal muscle

We examined the effects of undernutrition on muscle development during the first postnatal week in pigs. Eighteen piglets were subjected to three nutritional levels (300, 200 or 100 g/(kg body. d) of colostrum then milk) between birth and slaughter at 7 d of age. Longissimus lumborum (LL), a fast-twitch glycolytic muscle, and rhomboideus (RH), a mixed slow- and fast-twitch oxido-glycolytic muscle, were taken for myofiber typing and biochemical analyses. Enzyme activities of lactate dehydrogenase (LDH), citrate synthase (CS) and beta-hydroxy-acyl-CoA-dehydrogenase (HAD) were used as markers of glycolytic, oxidative and lipid beta-oxidation capacities, respectively. Undernutrition selectively decreased (P < 0.001) hypertrophy of the future fast-twitch glycolytic fibers in LL. Contractile and metabolic maturation was delayed in the later maturing LL, as reflected by a decrease in muscle protein concentration (P < 0.01), an increase (P < 0.05) in the percentage of myofibers still expressing the fetal myosin heavy chain (MyHC), a lower postnatal increase in LDH activity (P < 0.001) and a delayed decrease in the percentage of IIa MyHC positive fibers (P < 0.001). Otherwise, restriction tended (P < 0.10) to increase the percentage of slow type I MyHC containing fibers in both muscles and of alpha-cardiac MyHC positive fibers in RH (P < 0.05). The LDH/CS ratio decreased dramatically (P < 0.001) after restriction, to a greater extent in LL than in RH. These changes denoted a more oxidative metabolism using fewer carbohydrates and more lipids in restricted pigs, as suggested by the increased activity of HAD (P < 0.001) and decreased respiratory quotient (P < 0.001).     

Development of a xenogeneic DNA vaccine program for canine malignant melanoma at the Animal Medical Center

INTRODUCTION: Canine malignant melanoma (CMM) is an aggressive neoplasm treated with surgery and/or fractionated RT; however, metastatic disease is common and chemoresistant. Preclinical and clinical studies by our laboratory and others have shown that xenogeneic DNA vaccination with tyrosinase family members can produce immune responses resulting in tumor rejection or protection and prolongation of survival. These studies provided the impetus for development of a xenogeneic DNA vaccine program in CMM. MATERIALS AND METHODS: Cohorts of three dogs each received increasing doses of xenogeneic plasmid DNA encoding either human tyrosinase (huTyr; 100/500/1500 mcg), murine GP75 (muGP75; 100/500/1500 mcg), murine tyrosinase (muTyr; 5 dogs each at 100/500 mcg), muTyr+/-HuGM-CSF (9 dogs at 50 mcg muTyr, 3 dogs each at 100/400/800 mcg HuGM-CSF, or 3 dogs each at 50 mcg muTyr with 100/400/800 mcg HuGM-CSF), or 50 mcg MuTyr intramuscularly biweekly for a total of four vaccinations. RESULTS: The Kaplan-Meier median survival time (KM MST) for all stage II-IV dogs treated with huTyr, muGP75 and muTyr are 389, 153 and 224 days, respectively. Preliminarily, the KM MST for stage II-IV dogs treated with 50 mcg MuTyr, 100/400/800 mcg HuGM-CSF or combination MuTyr/HuGM-CSF are 242, 148 and >402 (median not reached) days, respectively. Thirty-three stage II-III dogs with loco-regionally controlled CMM across the xenogeneic vaccine studies have a KM MST of 569 days. Minimal to mild pain was noted on vaccination and one dog experienced vitiligo. We have recently investigated antibody responses in dogs vaccinated with HuTyr and found 2- to 5-fold increases in circulating antibodies to human tyrosinase. CONCLUSIONS: The results of these trials demonstrate that xenogeneic DNA vaccination in CMM: (1) is safe, (2) leads to the development of anti-tyrosinase antibodies, (3) is potentially therapeutic, and (4) is an attractive candidate for further evaluation in an adjuvant, minimal residual disease Phase II setting for CMM.     

棕腹刺豚肌肉营养成分与品质的评价

[目的]评价棕腹刺豚（L.wheeleri）肌肉营养成分与品质,为棕腹刺豚的利用提供科学依据。[方法]参照食品检验国标方法测定5尾棕腹刺豚肌肉的营养成分。蛋白质营养价值评价采用氨基酸比值系数法,以W HO/FAO氨基酸参考模式为评价标准,并与全鸡蛋蛋白质进行对照比较。[结果]棕腹刺豚肌肉中粗蛋白质量分数19.30%,粗脂肪质量分数1.94%,水分质量分数78.50%,碳水化合物质量分数0.26%。肌肉中含有18种氨基酸,总量为18.49%（质量分数,鲜样）,其中9种人体必需氨基酸占氨基酸总量的44.52%,必需氨基酸的构成比例基本符合W HO/FAO的标准。棕腹刺豚的第一限制性氨基酸为含硫氨基酸（Met＋Cys）,氨基酸比值系数分为71.00,5种鲜味氨基酸占氨基酸总量的44.86%。[结论]棕腹刺豚有较高的食用价值与保健作用。     

白细胞介素33在妊娠相关疾病中的作用

白细胞介素33（IL-33）属于IL-1β家族,是新发现的核定位因子,具有抗炎、促炎双重效应。IL-33可通过参与调节母胎免疫耐受过程以维持正常妊娠,同时,IL-33在多种妊娠相关疾病中发挥重要作用,如妊娠早期IL-33的表达异常或功能障碍可导致流产;胎盘中IL-33降低致使滋养细胞功能障碍,与子痫前期患者胎盘功能不良有关;妊娠中晚期蜕膜中IL-33表达降低或功能障碍在早产的发生发展中发挥重要作用;IL-33可触发多种细胞的钙通道增加细胞内的钙离子水平,且已发现钙离子在子宫平滑肌收缩中发挥重要作用,但是关于IL-33在子宫平滑肌的研究尚待深入。而早产的发生最终都伴随着子宫平滑肌收缩, 故探讨IL-33在妊娠相关疾病以及平滑肌细胞收缩中的作用,有助于进一步研究早产的发生发展机制,从而为早产的诊治提供新思路。     

骨髓间充质干细胞移植治疗帕金森病的研究进展

骨髓间充质干细胞是一类具有多向分化潜能的成体干细胞,它可在体外分离、培养、扩增及诱导分化,目前可分化为骨细胞、软骨细胞、肌细胞、神经细胞等,是组织工程的重要种子细胞之一。骨髓间充质干细胞转化为神经细胞并进行细胞替代或转基因治疗这一方法为神经系统退行性疾病的治疗提供了广阔的前景。     

间充质干细胞治疗心脏疾患的研究进展

间充质干细胞(mesenchymal stem cells,MSCs)存在于多种组织内,是一种具有自我增殖和多项分化潜能的成体干细胞。MSCs体外易培养,且具有免疫调节特性,使其成为实现组织再生的种子细胞。动物心肌梗死模型证实MSCs体内移植,能够分化为心肌细胞和血管细胞,募集内源性心肌干细胞,分泌系列细胞因子,这些特性能够防止和逆转心室重构。Ⅰ期临床试验表明MSCs移植具有提高左室射血分数、抑制心室重构、减少瘢痕面积的作用。     

Birth and clinical pregnancy from fresh and frozen oocytes fertilized with cryopreserved testicular spermatozoa

This is the first report showing a second clinical pregnancy of a couple who already have a baby from a previous frozen embryo transfer cycle when the embryos were generated from fresh oocytes that were fertilized by intracytoplasmic sperm injection (ICSI) using frozen testicular spermatozoa (the couple have unsuccessful fresh and frozen embryo transfer cycles). Fifty-two months after the first IVF/ICSI cycle the couple had their second IVF/ICSI cycle, but the collected oocytes (n=8) were frozen because no spermatozoa was obtained from the frozen testicular tissue samples which were cryopreserved prior to the first IVF/ICSI cycle. New testicular tissue samples were obtained and frozen. Finally, 58 months after the first IVF/ICSI cycle all of the 8 frozen oocytes of the couple were thawed and fertilized by ICSI using frozen testicular spermatozoa obtained from the newly cryopreserved testicular tissue. Three embryos were transferred and the couple has an ongoing pregnancy, which is in the 20(th) week of pregnancy. Our case report shows that: 1) developmentally competent embryos can be generated by ICSI of frozen-thawed testicular spermatozoa into both fresh and frozen human oocytes, and 2) clinical pregnancy and a healthy baby can be conceived from both frozen and fresh oocytes fertilized with cryopreserved testicular spermatozoa.     

Coenzyme Q10 Supplementation Increases Removal of the ATXN3 Polyglutamine Repeat,Reducing Cerebellar Degeneration and Improving Motor Dysfunction in Murine Spinocerebellar Ataxia Type 3

Coenzyme Q10 (CoQ10), a well-known antioxidant, has been explored as a treatment in several neurodegenerative diseases, but its utility in spinocerebellar ataxia type 3 (SCA3) has not been explored. Herein, the protective effect of CoQ10 was examined using a transgenic mouse model of SCA3 onset. These results demonstrated that a diet supplemented with CoQ10 significantly improved murine locomotion, revealed by rotarod and open-field tests, compared with untreated controls. Additionally, a histological analysis showed the stratification of cerebellar layers indistinguishable from that of wild-type littermates. The increased survival of Purkinje cells was reflected by the reduced abundance of TUNEL-positive nuclei and apoptosis markers of activated p53, as well as lower levels of cleaved caspase 3 and cleaved poly-ADP-ribose polymerase. CoQ10 effects were related to the facilitation of the autophagy-mediated clearance of mutant ataxin-3 protein, as evidenced by the increased expression of heat shock protein 27 and autophagic markers p62, Beclin-1 and LC3II. The expression of antioxidant enzymes heme oxygenase 1 (HO-1), glutathione peroxidase 1 (GPx1) and superoxide dismutase 1 (SOD1) and 2 (SOD2), but not of glutathione peroxidase 2 (GPx2), were restored in 84Q SCA3 mice treated with CoQ10 to levels even higher than those measured in wild-type control mice. Furthermore, CoQ10 treatment also prevented skeletal muscle weight loss and muscle atrophy in diseased mice, revealed by significantly increased muscle fiber area and upregulated muscle protein synthesis pathways. In summary, our results demonstrated biochemical and pharmacological bases for the possible use of CoQ10 in SCA3 therapy.     

人脐血间充质干细胞分离培养方法的优化

目的探索脐血来源的间充质干细胞体外分离培养的更优方法。方法无菌条件下抽取正常足月剖宫产胎儿的脐带血,肝素抗凝,用淋巴细胞分离液分离脐血单个核细胞。50份脐血分为对照组和改良组,每组25份,分别以1.5×10^7/ml的细胞密度接种于25 cm^2培养瓶中。对照组贴壁3 d后弃上清,去除未贴壁细胞,更换新鲜培养液;改良组贴壁0.5 h后弃上清,去除未贴壁细胞,更换新鲜培养液。结果对照组杂细胞较多,最终成功培养出8份脐血间充质干细胞;改良组杂细胞较少,最终成功培养出18份脐血间充质干细胞。经流式细胞仪检测脐血间充质干细胞的免疫表型,结果显示,所培养出的脐血间充质干细胞不表达或极弱表达CD34、CD106等造血细胞标志,稳定地高表达CD29、CD44、CD105等间充质细胞相关的表面抗原标记物。结论采取脐血单个核细胞贴壁0.5 h弃上清更换新鲜培养液可以明显提高脐血间充质干细胞的培养成功率,其结果优于脐血单个核细胞贴壁3 d弃上清更换新鲜培养液。