抗原呈递细胞外质体的研究进展

抗原呈递细胞(APC)在胞吐过程中,向细胞外分泌一种称为exosome的膜性小泡,含有丰富的MHC 分子和共刺激分子等,可以作为MIHC-肽复合物的运输工具,在细胞间传递抗原信息。经肿瘤抗原冲击树突细胞所 释放的外质体能直接或间接地刺激T细胞,从而产生强大的抗肿瘤免疫应答,有望成为新的非细胞免疫治疗载体。

Skin care efficacy study of recombinant humanized collagen based on in vitro level

Studying the skin care efficacy of recombinant humanized collagen based on in vitro level.The stability of the recombinant humanized collagen was first analyzed by treating at different temperatures,then its skincare efficacy based on in vitro level was evaluated by detecting the inhibition rate of elastase,the inhibition rate of collagenase,the protein content of type I collagen in human fibroblasts,the inhibition of reactive oxygen species(ROS)with human keratinocytes,and the effects of the recombinant humanized collagen on the expression of hyaluronic acid(HA),filaggrin(FLG)and transglutaminase 1(TGM1)in keratinocytes.The results showed that recombinant humanized collagen was able to maintain stability at temperatures below 70℃.With regard to its skincare efficacy,recombinant humanized collagen could inhibit elastase and collagenase activities and promote the increase of type I collagen content in human fibroblasts.It also showed good inhibition of ROS in keratinocytes in vitro and could increase the expression of HA,FLG,and TGM1 in keratinocytes.In short,the recombinant humanized collagen exhibited a favourable skin care effect in vitro level.This study proved that it has potential firming,anti-wrinkle,moisturizing,and repairing efficacy,and is a valuable cosmetic raw material.

Improvement of in vitro Cytoplasmic Maturation of Denuded Porcine Oocytes by Coculture with Follicular Mural Granulosa Cells

[Objective] This study aimed to improve the in vitro maturation quality of denuded porcine oocytes and provide scientific basis for establishing a stable and efficient denuded oocyte culture system. [Method] The first polar body extrusion rate, oocyte glutathione （GSH） content, positive rate of brilliant cresyl blue （BCB） staining and development potential of activated oocytes or fertilized oocytes were employed as main indicators to investigate the effects of follicular mural granulosa cell （MGC） coculture on cytoplasmic maturation of cumulus cell-removal oocytes （Denuded Oocyte, DO）. [Result] According to in vitro maturation results, compared with DO group, the first polar body extrusion rate of porcine oocytes in DO＋MGC group was not significantly different, but the nuclear maturation process was improved and was more similar to that in COC （cumulus-oocyte complex） group. Detection of GSH content in mature oocytes showed that there was no significant difference between DO＋ MGC group （optical density of 1 053.67） and COC group （optical density of 1 426.00） or between DO＋MGC group and COC＋GC group （optical density of 1 541.00）, however, GSH content in mature oocytes of DO group （optical density of 724.67） was significantly lower than that of COC group and COC＋GC group （P0.05）. Detection of glucose-6-phosphate dehydrogenase （G6PDH） activity showed that there was no significant difference in BCB positive oocyte rate between DO ＋MGC group （88.26% ） and COC group （92.75%） or between DO＋MGC group and DO group （82.86% ）, however, BCB positive oocyte rate of DO group was significantly lower than that of COC group （P0.05）. Furthermore, the cleavage rate and blastocyst rate of activated mature oocytes derived from DO ＋MGC group （94.98% and 43.67% , respectively） were significantly higher than those from DO group （52.54% and 8.97%, respectively） （P0.05）, and were not significantly different compared with those from COC group （97.11% and 38.30%, respectively）. In addition, the cleavage rate of fertilized oocytes derived from DO＋MGC group （72.65%） showed no significant difference compared with that from DO group （63.59%）, but the blastocyst rate of DO＋MGC group was significantly higher than that of DO group （9.88%） （P0.05）. [Conclusion] MGC coculture can significantly improve the in vitro cytoplasmic maturation quality of denuded porcine oocytes, thereby enhancing the subsequent developmental potential.

Production of Somatic Hybrid Plants Between Two Types of Wheat Protoplasts and the Protoplasts of Haynaldia villosa

小麦 (TriticumaestivumL .)济南 177的两种原生质体 ,一种来自快速生长的悬浮细胞 ,它们因长期继代而丧失分化能力 ,其染色体只有 2n =2 4～ 2 8;另一种来自可以再生的愈伤组织 ,其原生质体不能持续分裂。它们中任一种与UV照射过的簇毛麦原生质体融合均不能再生植株。然而当它们混合在一起作为受体时 ,能够获得再生绿色植株。细胞核基因和胞质基因的分析证明这些绿色植株是杂种。以上事实说明这两种原生质体在融合时存在某些互补的关系 ,讨论了这种融合方式的可能作用及重要性。

Detachment of esophageal carcinoma cells from extracellular matrix causes relocalization of death receptor 5 and apoptosis

AIM： To investigate the effect of detachment of esophageal cancer cells from extracellular matrix on the localization of death receptor 5 （DR5） and apoptosis. METHODS： Anchorage-dependent EC9706 cells of esophageal squamous cell carcinoma were pretreated or not treated with brefeldin A. Detached cells were harvested by ethylenediaminetetraacetic acid digestion. Expression and localization of DR5 in these cells were determined by immunocytochemical and immunofluorescence assays, as well as flow cytometry analysis. Apoptosis of EC9706 cells was detected by flow cytometry after stained with fluorescein isothiocyanate-labeled annexin V/propidium iodide. Activation of caspase 8 was detected by Western blot analysis. RESULTS： Immunocytochemical assay indicated that DR5 was predominantly perinuclear in adherent cells but was mainly localized in cell membrane in detached cells. In addition, immunofluorescence assay also confirmed the above-mentioned results, and further demonstrated that DR5 was present in the form of coarse granules in detached cells, but in the form of fine granules in adherent cells. Cytometry analysis revealed higher levels of DR5 expression on the surfaces of brefeldin-A-untreated cells than on the surfaces of brefeldin-A-treated cells, but brefeldin A treatment did not affect the total DR5 expression levels. Moreover, nocodazole did not influence the extracelluar DR5 expression levels in EC9706 cells. Apoptosis assay revealed that detached cells were more sensitive to DR5 antibody-induced apoptosis than adherent ceils. Western blotting showed that caspase 8 was activated in temporarily detached cells 4 h earlier than in adherent cells. CONCLUSION： Progress from adhesion to detachment of EC9706 cells causes DR5 relocalization, and promotes cytoplasmic translocation of DR5 to cell surfaces via a Golgi-dependent pathway. Moreover, it might also result in DR5 aggregation to render apoptosis of detached cells.

Thyroid hormone regulation of apoptotic tissue remodeling during anuran metamorphosis

Anuran metamorphosis involves systematic transformations of individual organs in a thyroid hormone (TH)-dependent manner. Morphological and cellular studies have shown that the removal of larval or- gans/tissues such the tail and the tadpole intestinal epithelium is through programmed cell death or apop- tosis. Recent molecular investigations suggest that TH regulates metamorphosis by regulating target gene expression through thyroid hormone receptors (TRs), which are DNA-binding transcription factors. Cloning and characterization of TH response genes show that diverse groups of early response genes are induced by TH. The products of these TH response genes are believed to directly or indirectly affect the expression and/or functions of cell death genes, which are conserved at both sequence and function levels in different animal species. A major challenge for future research lies at determining the signaling pathways leading to the activation of apoptotic processes and whether different death genes are involved in the regulation of apoptosis in different tissues/organs to effect tissue-specific transformations.

The matrix metalloproteinase stromelysin-3 cleaves laminin receptor at two distinct sites between the transmembrane domain and laminin binding sequence within the extracellular domain

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. However like other MMPs, the molecular basis of ST3 function in vivo remains unclear due to the lack of information on its physiological substrates. Furthermore, ST3 has only weak activities toward all tested ECM proteins. Using thyroid hormone-dependent Xenopus laevis metamorphosis as a model, we demonstrated previously that ST3 is important for apoptosis and tissue morphogenesis during intestinal remodeling. Here, we used yeast two-hybrid screen with mRNAs from metamorphosing tadpoles to identify potential substrate of ST3 during development. We thus isolated the 37 kd laminin receptor precursor (LR). We showed that LR binds to ST3 in vitro and can be cleaved by ST3 at two sites distinct from where other MMPs cleave. Through peptide sequencing, we determined that the two cleavage sites are in the extracellular domain between the transmembrane domain and laminin binding sequence. Furthermore, we demon strated that these cleavage sites are conserved in human LR. These results together with high levels of human LR and ST3 expression in carcinomas suggest that LR is a likely in vivo substrate of ST3 and that its cleavage by ST3 may alter cell-extracellular matrix interaction, thus, playing a role in mediating the effects of ST3 on cell fate and behavior ob- served during development and pathogenesis.

Association of DNA with nuclear matrix in in vitro assembled nuclei induced by rDNA from Tetrahymena shanghaiensis in Xenopus egg extracts

The nuclei assembled from exogenous DNA or chromatin in egg extracts resemble their in vivo counterparts in many aspects. However, the distribution pattern of DNA in these nuclei remains unknown. We introduced rDNA from the macronuclei of Tetrabymena into Xenopus cellfree extracts to examine the association of specific DNA sequences with nuclear matrix (NM) in the nuclei assembled in vitro. Our previous works showed the 5’NTS (nontranscription sequences) of the rDNA specifically bind to the NM system in the macronuclei. We show now the rDNA could induce chromatin assembly and nuclear formation in Xenopus cell-free system. When we extracted the NM system and compared the binding affinity of different regions of rDNA with the NM system, we found that the 5’NTS still hold their binding affinity with insoluble structure of the assembled nuclei in the extracts of Xenopus eggs.

Study on Apoptosis-Inducing Effect of XIAP Antisense Oligonucleotides on Glioblastoma Cells in Vitro

OBJECTIVE To investigate the apoptosis-inducing effect of XIAP antisense oligonucleotides on glioblastoma cells in vitro. METHODS There were 4 groups in our experiment. Group A, as a cell control group, had normal cell culture and no treatment applied. Group B, as a blank control group, had normal cell culture and no liposome control of ASODN. Group C was N-ODN. Group D was the ASODN group. RT-PCR and Western blot assay were conducted to detect the expression of XIAP in all A-172 ceil groups after treatment with XIAP antisense oligonucleotides （ASODN）. MTT assay and flow-cytometry （FCM） detection were used to detect the ability of cell anchoring growth and apoptotic rates of all groups. The processing time was 72 h. RESULTS The expression of XIAP in the A-172 cells was greatly down-regulated, after treated with XIAP-ASODN. Among different concentrations of ASODN, the 300nM was the most optimal one. The down-regulation of XIAP obviously inhibited the succinate dehydrogenase （SDH） activity of the A-172 cells and the increased apoptotic rate of A-172 cells （87.45%） was significantly higher than that of the A-172 in the control groups. There was a statistically significant difference between the treatment and control groups （P 〈 0.01）. CONCLUSION The XIAP-ASODN can effectively regulate the expression of the XIAP down, as a result, inhibit the growth of the glioblastoma cells （A-172） and obviously increase the apoptotic rate of the A-172 cells. The results killing role of XIAP-ASODN to the of the study manifest an overt glioblastoma cells.

Effects of connective tissue growth factor antisense oligonucleotides on the proliferation and collagen synthesis of the cultured human keloid fibroblasts in vitro

Objective: To explore the effects of connective tissue growth factor (CTGF) on the pathogenesis of human keloid. Methods: CTGF antisense oligonucleotides (ASODN) conjugated with isothiocyananate fluorescence was encapsulated by liposome, and then added into the human keloid fibroblasts (HKFs) culture media. The intracellular distribution of CTGF ASODN was observed by fluorescence microscopy in the fixed HKFs. The proliferation of HKFs was measured by MTT test. The collagen synthesis of HKFs was measured by 3H-proline incorporation method. Results: Compared with control group, the CTGF ASODN can inhibit the proliferation and collagen synthesis of the HKFs (P<0.01). Conclusion: CTGF ASODN has anti-fibrotic effects on keloid in vitro, and CTGF play an important role in promoting the fibrosis of keloid.

The Influence of Glucose on Numerical Simulation of a Vascular Solid Tumor Growth

Glucose is the mainly nutrient substances in tumor growth,which played an important role in tumor cells' growth,proliferation and immigration.Numerical simulation will help a good understanding for the influence of glucose which affected on a vascular solid tumor growth.We present a hybrid on-Lattice Model to simulate the influence of glucose on a-vascular tumor growth.The hybrid model we developed focuses on five key variables implicated in the invasion process:tumor cells,extracellular matrix,matrix-degradative enzymes,oxygen and glucose.And about the discrete model,we consider cell evolution dynamics on cell level.Results indicate that the number of proliferation and quiescent cells is decreasing by decreasing the initial glucose concentration,consequently increase necrotic area relatively.Thus there is inhabitation effect on tumor growth by decreasing initial glucose concentration.

Effects of intermittent negative pressure on osteogenesis in human bone marrow-derived stroma cells

Objective： We investigated the effects of intermittent negative pressure on osteogenesis in human bone marrowderived stroma cells （BMSCs） in vitro. Methods： BMSCs were isolated from adult marrow donated by a hip osteoarthritis patient with prosthetic replacement and cultured in vitro. The third passage cells were divided into negative pressure treatment group and control group. The treatment group was induced by negative pressure intermittently （pressure： 50 kPa, 30 min/times, and twice daily）. The control was cultured in conventional condition. The osteogenesis of BMSCs was examined by phase-contrast mi- croscopy, the determination of alkaline phosphatase （ALP） activities, and the immunohistochemistry of collagen type 1. The mRNA expressions of osteoprotegerin （OPG） and osteoprotegerin ligand （OPGL） in BMSCs were analyzed by real-time polymerase chain reaction （PCR）. Results： BMSCs showed a typical appearance of osteoblast after 2 weeks of induction by intermittent negative pressure, the activity of ALP increased significantly, and the expression of collagen type I was positive. In the treatment group, the mRNA expression of OPG increased significantly （P〈0.05） and the mRNA expression of OPGL decreased significantly （P〈0.05） after 2 weeks, compared with the control. Conclusion： Intermittent negative pressure could promote osteogenesis in human BMSCs in vitro.

Osteogenic potential of rabbit marrow stromal stem cells cultured in vitro: a hi stochemical and scanning electron microscopic study

To further investigate the osteogen ic potential of rabbit marrow stromal stem cells cultured in vitro. Methods: Rabbit marrow stromal stem cells were isolated by dens ity gradient centrifugation method and amplified in the flasks, using the osteog enic inducing conditions (OGC) as the culture media. The osteogenic potential of marrow stromal stem cells were investigated by means of bone seeking fluoresce nce (tetracycline) labelling, Alizarin red S (ARS) staining, Alcian blue Sirius red (AS) staining, and scanning electron microscope. Results: After being passaged, the marrow stromal stem cells in creased in number, became confluent and formed multi layer structure. The strom al stem cells excreted innumerable tiny granules, heaping up on the cell body an d merging gradually into foggy substances. These foggy substances kept on enlarg ing and formed round, oval, or flake like nodules. These nodules revealed brigh t golden yellow fluorescence under fluorescence microscope when labelled with te tracycline. Histochemical study with specific new bone staining with ARS reveale d positive calcium reaction, both denoting that they were newly formed bone tiss ues. After they were stained with AS, collagen and acid mucopolysaccharide were shown. Under scanning electron microscope, three types of cells with different c onfigurations were found. They were globular cells, spindle shaped cells and po lygonal or polygonal cells. Granules were excreted from the cells and heaped up on the cell body. Needle shaped and irregularly rectangular crystals also appea red and agglomerated with the granules to form nodules and trabecula like or fl ake like structures.Conclusions: Sequence of events of bone formation by rabbit mar row stromal stem cells cultured in vitro is fully depicted and confirmed, which provides the foundation for further investigating the mechanisms of osteoblast d ifferentiation from marrow stromal stem cells and the possible application in or thopaedics.

Salusins protect myocardium against ischemic injury by alleviating endoplasmic reticulum stress

Salusins are regulatory peptides that affect cardiovascular function. We previously reported that salusin-a and -β protected cultured cardiomyocytes from serum deprivation-induced cell death through upregulating glucose-regulated protein 78 （GRP78）, an endoplasmic reticulum （ER） resident protein whose overexpression acts as a marker and suppressor of ER stress. The present study examined whether salusin-α and -β inhibit ER stress in ischemic myocardium. In a rat model of myocardial infarction created by ligating the left anterior descending coronary artery （LAD）, salusin-α or -β was intravenously injected at 5 or 15 nmol kg-1 15 min prior to 2 h of LAD occlusion. The high dose of salusin-α and -β3 significantly improved heart function and hemodynamics in LAD-occluded rats, but had no effects in sham-operated rats. The arrhythmias caused by LAD oc- clusion were markedly attenuated by salusin-α and -β. The apoptotic rate in ischemic myocardium was reduced from 31.5%±3.7% to 19.8%±2.2% and 12.3%±2.2%, and the infarct size was reduced from 53.4%±4.0% of the risk area to 26.5%±9.7% and 23.7%±8.9% by 15 nmol kg-1 salusin-α and -β, respectively. Furthermore, salusin-α and -β prevented the ac- tivation of GRP78 and ER stress-specific apoptotic effectors caspase-12 and CHOP （C/EBP homologous protein）, and attenu- ated the reduction of an ER stress-associated antiapoptotic protein Bcl-2 in ischemic cardiac tissue. The salusins also inhibited the ER stress induced by tunicamycin in cultured rat H9c2 cardiomyocytes. These results indicate that salusins protect myo- cardium against ischemic injury by inhibiting ER stress and ER stress-associated apoptosis.

Effect of soothing liver therapy on oocyte quality and growth differentiation factor-9 in patients undergoing in vitro fertilization and embryo transfer

OBJECTIVE:To investigate the effect of Soothing liver therapy on infertile women undergoing in vitro fertilization and embryo transfer(IVF-ET)and to explore its mechanism.METHODS:Fifty-eight women with tubal infertility were randomized into two groups:30 in an experimental group treated with Xiaoyao powder(Shugan)plus gonadotropin-releasing hormone analog(GnRHa)/follicle-stimulating hormone(FSH)/human chorionic gonadotropin(hCG)and 28 in the control group who were treated with GnRHa/FSH/hCG only.The total gonadotropin(Gn)doses required,endometrial thickness,oocyte numbers,high quality embryo production rate and pregnancy rate of the two groups were compared.The concentration of growth differentiation factor-9(GDF-9)in follicular fluid was detected by western blotting and the expression of GDF-9 mRNA in granulosa cells was measured using reverse tran-scription-polymerase chain reaction amplification.RESULTS:In the experimental group,the Gn dose was significantly lower than that in the control group;the endometrial thickness,high quality embryo production and pregnancy rates were significantly higher and the expression of GDF-9 mRNA was also significantly higher than in the control group(all P<0.05).CONCLUSION:Shugan treatment can improve the pregnancy rate of women with tubal infertility;its mechanism is possibly related to the increased expression of GDF-9 in granulosa cells.

Biocompatibility Evaluation of Polyethylene Terephthalate Artificial Ligament Coating Hydroxyapatite by Fibroblasts Cells in Vitro

Hydroxyapatite(HA) based materials have been widely used in the field of ligament tissue engineering in the past decades.It has been previously reported that HA can increase the penetration of marrow-derived mesenchymal stem cells(MSCs) and MSCs cells into scaffolds due to increased cell differentiation in biological media.Additionally,it was found that there are much difference between MSCs and anterior cruciate ligament (ACL) cells.For that reason,we mainly evaluate the biocompatibility of polyethylene terephthalate(PET) silk scaffold with fibroblasts cells in vitro.We cultured mouse fibroblasts cells on the substrate of PET fiber and PET-HA scaffold,respectively,and then observed the morphology by using scanning electron microscopy.Our data indicate that PET-HA scaffold has good biocompatibility with fibroblasts cells and can potentially be useful in enhancing the fibroblasts cell differentiation and proliferation.