放射诱导调控序列的合成及其辐射诱导特性的研究

目的　合成放射诱导调控序列并鉴定其辐射诱导特性。方法　利用人工寡核苷酸片段合成含有 6个重复CArG元件的放射诱导调控序列 ,以绿色荧光蛋白 (GFP)作为报告基因转染Tca8113细胞检测其辐射诱导特性。结果　低剂量放射线照射可诱导这种人工调控序列增强GFP在Tca8113细胞中的表达 ,且 3Gy剂量最为明显 ,提高到放射前的 16 1%。结论　人工合成放射诱导调控序列在低剂量放射线照射下能明显增强其下游外源性基因的表达 ,为进一步研究放射 -基因治疗奠定了基础。     

Ad-vcam-1-gfp重组腺病毒转染人脐血源基质细胞的实验研究

本研究探讨血管细胞黏附分子（vcam-1）修饰的人脐血源基质细胞（human umbilical cord blood derived strom cells，CBDSC）在造血调控中的作用。采用DNA重组技术，将目的基因vcam-1克隆至含有报告基因gfp的穿梭质粒；在BJ5183细胞中与pAdeasy—1质粒进行同源重组，产生重组腺病毒载体转染人脐血源基质细胞并进行鉴定。结果表明：vcam-1基因与pAdTrack—CMV载体成功连接，经NotⅠ／XhoⅠ双酶切后电泳可见2个大小约为9kb和2kb左右条带，经PCR法扩增后电泳可见1个600bp左右条带，证明pAdTrack—CMV—vcam-1重组质粒构建成功。pAdTrack—CMV—vcam-1质粒与pAdeasy—1质粒同源重组后，产物经PACⅠ酶切后电泳可观察到2个大小约为31kb、4kb左右条带，与预期结果相符。腺病毒载体转染人脐血源基质细胞后免疫化学、RT—PCR、荧光显微镜等方法均检测到目的基因vcam-1的表达。结论：构建ad—vcam-1-gfP重组腺病毒载体可成功转染人脐血源基质细胞并使其vcam-1表达增高。     

Cytological Kinetics of Periodontal Ligament in an Experimental Occlusal Trauma Model

Using a model of experimental occlusal trauma in mice, we investigated cytological kinetics of periodontal ligament by means of histopathological, immunohistochemical, and photographical analysis methods. Periodontal ligament cells at furcation areas of molar teeth in the experimental group on day 4 showed a proliferation tendency of periodontal ligament cells. The cells with a round-shaped nucleus deeply stained the hematoxylin and increased within the day 4 specimens. Ki67 positive nuclei showed a prominent increase in the group on days 4 and 7. Green Fluorescent Protein (GFP) positivity also revealed cell movement but was slightly slow compared to Ki67. It indicated that restoration of mechanism seemed conspicuous by osteoclasts and macrophages from bone-marrow-derived cells for the periodontal ligament at the furcation area. It was suggested that the remodeling of periodontal ligament with cell acceleration was evoked from the experiment for the group on day 4 and after day 7. Periodontal ligament at the furcation area of the molar teeth in this experimental model recovered using the cells in situ and the bone-marrow-derived cells.     

糖尿病并发症相关基因醛糖还原酶酵母细胞模型的建立

目的 构建由绿色荧光蛋白（GFP）标记的糖尿病慢性并发症相关基因醛糖还原酶（AR）表达载体，并在酵母细胞中表达，建立AR抑制剂筛选模型。方法 构建AR与GFP的嵌合基因，将此AR：：GFP嵌合基因克隆到酵母表达载体pYEX-BX中，再将重组载体pYEX-BX-AR：：GFP转化至酵母宿主菌INVSC1，在SC-UD选择性培养基中表达AR：：GFP。结果 PCR扩增出约l106bp大小片段，为AR基因片段；RT-PCR在酵母细胞中检测到ARmRNA的表达；经CuS04诱导后可见AR：：GFP融合蛋白的绿色荧光；诱导剂CuSO4终浓度在100～200μmol／L范围内相对荧光强度较高，细胞密度适中；培养基SC-UD的pH值为6左右最适合细胞生长和荧光表达。结论 AR：：GFP在酿酒酵母中成功地表达，酵母细胞是研究目的基因功能与表达的良好模式菌。     

Allogeneic administration of fetal membrane-derived mesenchymal stem cells attenuates acute myocarditis in rats

We reported previously that the autologous administration of bone marrow-derived mesenchymal stem cells (BM-MSC) significantly attenuated myocardial dysfunction and injury in a rat model of acute myocarditis by stimulating angiogenesis and reducing inflammation. Because BM aspiration procedures are invasive and can yield low numbers of MSC after processing, we focused on fetal membranes (FMs) as an alternative source of MSC to provide a large number of cells. We investigated whether the allogeneic administration of FM-derived MSC (FM-MSC) attenuates myocardial injury and dysfunction in a rat myocarditis model. Experimental autoimmune myocarditis (EAM) was induced in male Lewis rats by injecting porcine cardiac myosin. Allogeneic FM-MSC obtained from major histocompatibility complex-mismatched ACI rats (5 × 10⁵ cells/animal) were injected intravenously into Lewis rats one week after myosin administration. At day 21, severe cardiac inflammation and deterioration of cardiac function were observed. The allogeneic administration of FM-MSC significantly attenuated inflammatory cell infiltration and monocyte chemoattractant protein 1 expression in the myocardium and improved cardiac function. In a T-lymphocyte proliferation assay, the proliferative response of splenic T lymphocytes was significantly lower in cells obtained from FM-MSC-treated EAM rats that reacted to myosin than in cells obtained from vehicle-treated rats with EAM. T-lymphocyte activation was significantly reduced by coculture with FM-MSC. The allogeneic administration of FM-MSC attenuated myocardial dysfunction and inflammation, and the host cell-mediated immune response was attenuated in a rat model of acute myocarditis. These results suggest that allogeneic administration of FM-MSC might provide a new therapeutic strategy for the treatment of acute myocarditis.     

Immunogenicity and safety of an E. coli-produced bivalent human papillomavirus (type 16 and 18) vaccine: A randomized controlled phase 2 clinical trial

BackgroundThis study aimed to investigate the dosage, immunogenicity and safety profile of a novel human papillomavirus (HPV) types 16 and 18 bivalent vaccine produced by E. coli.MethodsThis randomized, double-blinded, controlled phase 2 trial enrolled women aged 18–25 years in China. Totally 1600 eligible participants were randomized to receive 90 μg, 60 μg, or 30 μg of the recombinant HPV 16/18 bivalent vaccine or the control hepatitis B vaccine on a 0, 1 and 6 month schedule. The designated doses are the combined micrograms of HPV16 and 18 VLPs with dose ratio of 2:1. The immunogenicity of the vaccines was assessed by measuring anti-HPV 16 and 18 neutralizing antibodies and total IgG antibodies. Safety of the vaccine was assessed.ResultsAll but one of the seronegative participants who received 3 doses of the HPV vaccines seroconverted at month 7 for anti-HPV 16/18 neutralizing antibodies and IgG antibodies. For HPV 16, the geometric mean titers (GMTs) of the neutralizing antibodies were similar between the 60 μg (GMT = 10,548) and 90 μg (GMT = 12,505) HPV vaccine groups and were significantly higher than those in the 30 μg (GMT = 7596) group. For HPV 18, the GMTs of the neutralizing antibodies were similar among the 3 groups. The HPV vaccine was well tolerated. No vaccine-associated serious adverse events were identified.ConclusionThe prokaryotic-expressed HPV vaccine is safe and immunogenic in women aged 18–25 years. The 60 μg dosage formulation was selected for further investigation for efficacy.Clinical trials registration: NCT01356823.     

Participation of bone marrow-derived cells in hippocampal vascularization after status epilepticus

PurposeDiseases such as temporal lobe epilepsy, brain trauma and stroke can induce endothelial cell proliferation and angiogenesis in specific brain areas. During status epilepticus (SE), bone marrow-derived cells are able to infiltrate and proliferate, dramatically increasing at the site of injury. However, it is still unclear whether these cells directly participate in vascular changes induced by SE.MethodTo investigate the possible role of bone marrow-derived cells in angiogenesis after seizures, we induced SE by pilocarpine injection in previously prepared chimeric mice. Mice were euthanized at 8 h, 7 d or 15 d after SE onset.ResultsOur results indicated that SE modified hippocampal vascularization and induced angiogenesis. Further, bone marrow-derived GFP⁺ cells penetrated through the parenchyma and participated in the formation of new vessels after SE. We detected bone marrow-derived cells closely associated with vessels in the hippocampus, increasing the density of blood vessels that had decreased immediately after pilocarpine-induced SE.ConclusionWe conclude that epileptic seizures directly affect vascularization in the hippocampus mediated by bone marrow-derived cells in a time-dependent manner.     

增强型绿色荧光蛋白真核表达载体的构建和表达

目的　构建增强型绿色荧光蛋白(Enhanced green fluorescent protein, EGFP)编码基因的真核表达载体 pcDNA3.1(+) GFP,并观察其在Hep 2细胞中的表达情况。　方法　据已知的EGFP基因序列,设计合成 1 对引物,并引入Hind Ⅲ和EcoR Ⅴ酶切位点。应用PCR技术,从含有 EGFP的 pAdTrack CMV中扩增 EGFP编码基因。通过 TA连接将其克隆入pGEM T easy载体,经PCR及限制性内切酶鉴定后,插入真核表达质粒pcDNA3.1(+)中,转化Esche richia coli DH5α感受态细胞,于Amp+ LB平板上筛选阳性克隆。重组子经 Hind Ⅲ和EcoRⅤ双酶切、PCR鉴定,将该载体转染人喉癌细胞 Hep 2 后 48 h观察 EGFP表达情况。　结果　成功构建了含 EGFP 编码基因的真核表达载体pcDNA3.1(+) GFP,并成功转染Hep 2细胞,在倒置荧光显微镜下呈现绿色光。　结论　获得可产生绿色荧光的 EG FP,能方便地用作报告基因和筛选标记。