Localization of leptin and leptin receptor in the bovine adenohypophysis

The present study was carried out to detail the cellular localization of leptin (Lep) and the leptin receptor (LepR) in the bovine adenohypophysis. Lep immunoreactivity (Lep-ir) was found in about 30% of adenohypophysial cells in the gland. Immunochemistry of Lep and specific hormones using serial sections revealed that Lep-ir was present in 60.4% of somatotrophs, 15.9% of gonadotrophs, 6.5% of mammotrophs, 6.5% of thyrotrophs and 2.4% of corticotrophs. Both the common short isoform (OBRa) and the long isoform (OBRb) of LepR mRNA were expressed in the bovine adenohypophysis. LepR immunoreactivity (LepR-ir) was found in only 2.8% of the adenohypophysial cells and over 50% of LepR-ir cells were gonadotrophs, in which most of the cells were distributed in the zona tuberalis. The findings on Lep and LepR in the adenohypophysial cells indicate that Lep may regulate gonadotroph function through autocrine/paracrine pathway in the bovine adenohypophysis.     

Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model

Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.     

Flow-cytometric Studies of the Phagocytic Capacities of Equine Neutrophils

Methodological aspects of flow-cytometric evaluation of the phagocytic properties of equine neutrophils were elucidated. The kinetics of attachment and ingestion were studied, and the phagocytic process was more rapidly completed when serum-opsonized yeast cells were used than with use of IgG-opsonized yeast cells. Trypan blue was successfully used to quench fluorescence of non-ingested yeast cells. There were only minor differences in the kinetics of phagocytosis between quenched and un-quenched samples, indicating that attachment is rapidly followed by ingestion. Trypan blue quenching caused loss of cells with light scattering properties of granulocytes, although this did not affect the determined frequencies of truly phagocytic neutrophils. Aggregation of yeast cells proved to be a disturbance but not an obstacle to the determination of frequencies of actively phagocytic cells. Flow cytometry is well suited for studies of phagocytosis of yeast cells by equine neutrophils, and the trypan blue quenching provides a means of eliminating false-positive events due to aggregation of yeast cells. The main advantage of the flow-cytometric method is the possibility of rapid processing of a large number of samples, making the method useful for studies of herds.     

Research Advances in Escherichia coli K1 Penetration Across Blood-brain Barrier

E.coli K1 strain is a representative strain of neonatal sepsis and meningitis, which causes disease by blood circulation to the brain. The molecular mechanism of E.coli K1 that adhering and invading the brain microvascular endothelial cells (BMECs) and cross the blood brain barrier (BBB) has been focused on by many scholars. In this review, we focused on the gene regulation mechanisms and signaling pathway of E.coli K1 crossing the blood brain barrier to understand the molecular mechanism of the infection caused by E.coli and provide reference for prevention and treatment programs.     

Metastatic cancer of unknown primary in 21 dogs

The aim of this retrospective study was to describe clinical features, treatment and outcome of 21 dogs with metastatic cancer of unknown primary (MCUP), a biopsy‐proven malignancy being diagnosed at a metastatic stage, in which the anatomical origin of the primary tumour cannot be detected. All dogs underwent total‐body computed tomography. Signalment, type and duration of clinical signs, metastasis site, pathology results, treatment and outcome were recorded. Carcinoma was the most common diagnosis (57.1%), followed by sarcoma, melanoma and mast cell tumour. The median number of disease sites per dog was 2, with bones, lymph nodes, lungs and spleen being the most frequent metastatic locations. The median survival for all dogs was 30 days. Overall, a primary site was not identified in 20 (95.2%) dogs. MCUP encompasses a variety of different pathologic entities and harbours a poor prognosis.     

The stimulatory effect of TLRs ligands on maturation of chicken bone marrow-derived dendritic cells

Dendritic cells (DCs) are crucial for initiation of both innate and adaptive immune responses. TLR ligands combine with Toll-like receptors (TLRs) expressed on the DC surface and induce DC maturation. The potential effect of three types of TLR ligands (Bacillus subtilis (B. subtilis) spores, polyinosinic–polycytidylic acid and CpG oligodeoxynucleotides) on chicken bone marrow-derived DCs (chBM-DCs) maturation was studied. The chBM-DCs cultured in presence of recombinant chicken granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 displayed the typical morphology of DCs after 7 days of culture. These immature chBM-DCs up-regulated the expression of MHC-II and of the putative CD11c, but had yet low to moderate levels of the CD40 and CD86 co-stimulatory molecules. After stimulation by the TLR ligands, the chBM-DCs displayed a more mature morphologic phenotype, significantly increased the CD40 and CD86 cell surface expression levels and gained the ability to stimulate proliferation of naive T cells in the allogeneic mixed lymphocyte reaction, compared to the immature chBM-DCs. In conclusion, our data demonstrated that all three TLR ligands were strong stimuli for driving chBM-DCs maturation in vitro, with B. subtilis spores being the most efficient.     

犬造血干细胞移植的作用机制及应用研究进展

造血干细胞(hematopoietic stem cell, HSC)位于造血系统的顶端, 是造血系统中唯一具有多能性和自我更新能力的细胞, 可以分化为各种功能的血细胞, 维持血液系统的建立和动态平衡, 是目前研究最为透彻、临床应用最为成熟的成体干细胞。造血干细胞的这些重要特性, 使得基于造血干细胞在治病机制研究和临床应用中的研究取得了很大进展。以造血干细胞为基础的再生医学治疗是目前治疗恶性血液病和遗传病的首选方法, 如造血干细胞治疗犬白细胞黏附缺陷综合征、犬遗传性贫血、犬淋巴瘤、犬白细胞减少症、犬X连锁严重联合免疫缺陷病等, 但由于白细胞抗原匹配的供体稀缺、可获得的造血干细胞数量有限等原因, 无法满足临床需求, 因此, 如何通过体外培养获得满足临床需要的造血干细胞成为近年来学者们研究的热点问题。作者参照现有研究成果对造血干细胞的来源和体外分离培养、治病机制进行系统的描述, 并对造血干细胞移植治疗遗传性溶血性贫血、白细胞黏附缺陷综合征、淋巴瘤、白细胞减少症、X连锁严重联合免疫缺陷病的临床研究进行综述, 以期为今后将造血干细胞广泛应用于临床治疗提供参考依据。     

ST贴壁细胞悬浮驯化及应用

为将ST贴壁细胞通过自主驯化,使其能在ST-S细胞无血清培养基中悬浮生长且能稳定传代,在摇瓶中实现高密度生长,并应用于猪伪狂犬病毒(PRV)悬浮培养,经ST-A低血清培养基适应培养,对一株贴壁的ST细胞进行了无血清的全悬浮驯化,并将PRV在悬浮细胞中连续盲传培养。结果显示:ST悬浮细胞能在无血清培养基中传代,培养48 h至第3代后,所能达到的最终细胞密度为3.00×10^(6 )cells/mL以上;PRV连续培养至第5代,毒价可达到109.0 TCID50/mL。结果表明,用专用培养基可使ST贴壁细胞实现悬浮驯化,并可应用于PRV悬浮培养。本研究为获得高密度ST悬浮细胞和提高PRV增殖效率奠定了技术基础。     

Evaluation of adverse reactions in dogs following intravenous mesenchymal stem cell transplantation

Background

Recent studies have assessed the therapeutic potential and drawbacks of mesenchymal stem cells (MSCs). The adverse reactions of intravenous transplantation of bone marrow (BM)-derived MSCs were examined at varying doses and frequencies of administration.Nine healthy beagle dogs were purchased from a commercial laboratory. The dogs were distributed equally (n = 3 per group) and randomly into three groups. All dogs received allogeneic BM-derived MSCs: 2 × 10⁶ once (group A), 2 × 10⁷ once (group B), and 2 × 10⁶ for three consecutive days (group C). Various laboratory examinations, multi-detector computed tomography features and histopathology were evaluated to clarify the clinical and diagnostic features of adverse reactions of MSCs administration, prior to receiving MSCs (pre procedure) and on days 1, 3, and 7 post transplantation.

Results

Only one dog had clinical signs during and after MSCs transplantation. Dogs receiving 2 × 10⁶ MSCs showed increased numbers of lymphocytes but the total white blood cell counts were not elevated (P < 0.01). Multi-detector computed tomography (MDCT) revealed pulmonary parenchymal changes in one dog and histopathologic examination revealed pulmonary parenchymal edema and hemorrhage in four dogs. The presence of pulmonary thromboembolism was not detected in either examination.

Conclusions

We considered the presence of pulmonary edema and hemorrhage as possible adverse reactions after intravenous MSCs transplantation; however these results should be cautiously interpreted.     

转EGFP基因山羊克隆胚的发育

利用脂质体包裹含EGFP基因的质粒,并将之导入山羊乳腺上皮细胞,经G418筛选获得阳性细胞,以阳性细胞作为核供体,利用核移植技术构建转基因克隆胚。结果表明：用转基因山羊乳腺上皮细胞作为核供体,电融合法更适合构建转基因克隆胚。转基因山羊克隆胚体外培养最佳方案是用SOFaa培养液,在培养72 h后加入10%的正常山羊血清（Normal goat serum,NGS）。荧光显微镜下观察到转基因克隆胚中EGFP的表达,大部分克隆胚发育到8-16细胞以后的时期,绿色荧光蛋白才开始逐渐表达,随着发育时间的延长,绿色荧光蛋白的表达也逐渐增强。说明外源基因在胚胎早期发育阶段可以表达,EGFP可以作为报告基因来实现对外源基因整合及表达的监测。