Reproductive characteristics of transgenic (TG) chickens caping an exogenous gene

An exogenous gene (lacZ/MiwZ) introduced into the germinal crescent region (GCR) of avian embryos was con-firmed to be successfully transferred to the gonads via the primordial germ cells (PGCs). Following hatching, the chickswere raised until the stage of sexual maturation. The incorporation of MiwZ DNA was detected in male and female trans-genic chickens, respectively. The normal male and female transgenic birds were subjected to artificial insemination ac-cording to routine methods. Fertilized eggs obtained from female transgenic chickens were incubated for 72 h and the em-bryos removed from the yolk were examined by X-gal staining to detect the introduction of MiwZ in the offspring. As aresult, the expression of MiwZ was detected in the offspring. Furthermore, the presence of MiwZ in the extracts fromembryos was also detected by polymerase chain reaction (PCR) analysis. In male transgenic chickens, the presence of in-jected MiwZ in the extracts from sperm was also confirmed. The exogenous gene intro     

携带人衰变加速因子基因转基因小鼠的建立

目的 通过建立转人衰变加速因子（ＤＡＦ）基因小鼠,为研究异种器官移植的超急性排斥反应提供有效的研究手段。方法 采用受精卵显微注射技术,将人ＤＡＦ基因导入小白鼠受精卵的原核中,Ｄｏｔ－ｂｌｏｔ及Ｓｏｕｔｈｅｒｎ－ｂｌｏｔ杂交确定阳性围基因小鼠。以Ｎｏｒｈｅｒｎ杂交法检测人ＤＡＦ基因的表达情况。结果 基因注射后共生出小鼠２４只,４只为转基因小鼠,人ＤＡＦ基因在其中１只小鼠体内得到表达,结论 所导入ＤＡ     

外源性NGF对转基因CGRP干细胞移植治疗骨质疏松大鼠的影响

目的 研究外源性神经生长因子（nerve growth factor,NGF)对转基因降钙素基因相关肽（calcitonin gene related peptide, CGRP)骨髓干细胞（mesenchymal stem cells,MSCs)移植治疗骨质疏松大鼠的影响。方法 构建CGRP基因表达载体,转染制备转基因CGRP-MSCs靶细胞;60只雌性SD大鼠骨质疏松模型随机均分为空白对照组（A组）、单纯MSCs移植治疗组（B 组）、CGRP-MSCs移植治疗组（C组）,CGRP-MSCs + NGF移植组（D组）。在治疗lw、2w、3w后,实时定量荧光PCR和Western blot检测3个时间段每组大鼠股骨骨组织中CGRP基因和蛋白的相对表达水平,评价各组疗效。结果 与A组比较,3个时间点,C组和D组中CGRP表达水平均显著上调（P<0.01);治疗lw后,C组和D组上调水平差异明显（P<0.01),D组低于C 组;治疗2w后,C组和D组之间仍保持显著差异（P <0.05),D组仍低于C组;治疗3w后,C组和D组上调水平差异明显缩 小,二者差异无统计学意义（P>0.05)。CGRP基因和蛋白相对表达水平在各组检测结果差异一致,同上。结论 在转基因 CGRP-MSCs移植治疗骨质疏松大鼠中联合应用外源性NGF,无累计效应,并可能抑制大鼠股骨骨组织中CGRP的表达,表明外源NGF于转基因CGRP-MSCs移植治疗骨质疏松症中可能并无促进作用,至少在CGRP表达层面无促进作用,而单纯的转基因CGRP-MSCs移植治疗骨质疏松大鼠疗效优于联合应用外源性NGF治疗方案。     

Ginkgo biloba extract (EGb 761) as an antioxidant factor of exogenous origin

Extracted from Ginkgo biloba leaves, the EGb 761 has a great number of therapeutical properties and anti-stress effects, for example the antioxidant one. We estimated in our experiment the influence of a chronic treatment with EGb 761 on the concentration of the main product of lipidic degradation--malondialdehyde (MDA), in cerebral cortex, in diencephalon, thymus and gastric mucosa. The experiment was performed on Wistar rats, male, young. The MDA estimation was performed by the spectrocalorimetry. We have determined that the stress raised the MDA concentration in all examined structures with approximately 100% and 80% of animals manifested stress ulcer. The treatment with EGb 761 before a stress inhibits the post-stress growth of MDA concentration and the process of stress ulcer formation.     

Production of transgenic goat (Capra hircus) with human Granulocyte Colony Stimulating Factor (hG-CSF) gene in Brazil

In order to produce transgenic goats with hG-CSF, a total of 24 adult Saanen and 48 adult undefined breed goats were used as donors and recipients, respectively. Donors were estrus-synchronized with vaginal sponges and superovulated by a treatment with 200 mg FSH given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced by injecting 100 microg GnRH 36 h after sponge removal. The recipients also received an estrus synchronization treatment. Donors were mated with fertile Saanen bucks and, approximately 72 h after sponge removal, zygotes were recovered surgically by flushing oviducts. The recovered zygotes were briefly centrifuged to a reliable visualization of the pronuclei. The DNA construct containing hG-CSF gene flanked by goat and bovine alphas1-casein sequences was injected into pronuclei of 129 zygotes. The microinjected embryos (3-6 per female) were transferred to 27 recipients. Ten recipients became pregnant and 12 kids were born. One transgenic male founder was identified in the group of kids. This is the first report of a birth of a transgenic goat in Latin America.     

Correction of anemia in uremic rats by intramuscular injection of lentivirus carrying an erythropoietin gene

BACKGROUND: Anemia is an inevitable consequence of chronic renal failure. Gene therapy using lentiviral vector (LV) would be an effective tool to treat anemia associated with renal failure. METHODS: A LV carrying the erythropoietin (EPO) cDNA was administered to skeletal muscle of partially nephrectomized rats, which is a model of uremia. The red blood cell production and serum EPO levels were temporally monitored in these rats. Polymerase chain reaction assays were done to validate the presence of the LV in the experimental rats. RESULTS: After a single intramuscular injection of LV at a dose of 55 microg p24 Gag antigen (approximately 5 x 10(7) transducing units), blood hematocrit (Hct) levels increased and peaked at 3 weeks (47.8 +/- 4.2%, p < 0.01, n = 8) with the levels being maintained for at least 20 weeks (duration of study; 44.9 +/- 3.3%, p < 0.01, n = 3). The control rats receiving LV expressing lacZ had Hct levels of 36.9 +/- 4.1% (n = 8) at 3 weeks and 33.1 +/- 3.7% (n = 4) at 20 weeks, respectively. The serum EPO levels in the rats injected with the LV expression EPO significantly increased (p < 0.01) to 156.3 +/- 3.0 mU/ml compared to the control rats (63.9 +/- 1.7 mU/ml). Polymerase chain reaction analysis of the isolated genomic DNA from the LV-injected rats showed specific positive detection of the LV in only the skeletal muscle tissue at the site of injection, whereas the other tissues, including the liver, spleen, and kidney, were negative. CONCLUSIONS: This study demonstrates that intramuscular injection of LV can produce highly efficient and sustained EPO secretion in uremic rats, and suggests that this approach could be an effective tool to deliver secretable proteins at therapeutic levels in various animal disease models.     

Absence of proximal duct apoptosis in the ventral prostate of transgenic mice carrying the C3(1)-TGF-beta type II dominant negative receptor

BACKGROUND: Prostatic epithelial cells are sensitive to the inhibitory effects of TGF-beta. However, TGF-beta signaling in the prostate is dependent on androgenic status. Under the in vivo conditions, it is difficult to dissociate the effect of TGF-beta from that of androgen on the prostate. METHODS: The objective of the present study was to create and verify a transgenic mouse system in which epithelial cells of the ventral prostate are insensitive to the actions of TGF-beta. By using a modified prostate-specific promoter, C3(1), the TGF-beta dominant negative receptor is only expressed in the epithelial cells of the ventral prostate, and these cells are resistant to TGF-beta. Morphology of transgenic animal prostates was compared to wild-type animal prostates by immunohistochemistry and microscopy. RESULTS: The prostate of transgenic mice exhibited an abnormal morphology with multiple layers of epithelial cells lining the proximal ducts, in contrast to the simple cuboidal monolayer of cells seen in the normal prostate. This observation was accompanied by a loss of apoptosis in this region, as seen by TUNEL assay. There was no significant difference in serum levels of testosterone between the wild-type and transgenic animals. CONCLUSIONS: These results demonstrated that a loss of sensitivity to TGF-beta results in the accumulation of multiple layers of epithelial cells in the proximal region of the ventral prostate. This abnormal growth illustrates that TGF-beta plays an important role in regulating prostate growth. The current transgenic system can be used as an experimental model to study the functional role of TGF-beta in prostatic growth and function.     

ProtEx~(TM)外源蛋白修饰技术在移植免疫研究中的作用

目的 采用ProtEx~(TM)外源蛋白修饰技术将外源蛋白FasL修饰于靶细胞,观察其在移植免疫中的作用.方法 1.将嵌合蛋白FasL(SA-FasL)修饰于大鼠脾细胞,检测蛋白的表达效率及其对脾细胞表面免疫分子的影响;分别以近交系WF大鼠和ACI大鼠作为供者和受者,进行异位心脏移植,按围手术期注射供者细胞的不同将受者分为3组:(1)SA-FasL组(n=23):注射经SA-FasL修饰的供者脾细胞;(2)SA组(n=20):注经链霉亲和素蛋白(SA)修饰的供者脾细胞;(3)Control组(n=10):未注射任何细胞,作为空白对照.术后观察移植心的跳动情况,停止跳动为发生排斥反应,持续有力的跳动超过100d为移植心长期存活.2.以链脲佐菌素(STZ)静脉注射诱导C57BL/6小鼠产生糖尿病;将SA-FasL蛋白修饰于小鼠胰岛;以近交系BALB/c小鼠作为供者,糖尿病C57BL/6小鼠作为受者,进行胰岛移植,根据移植不同的供者胰岛将受者分为组:(1)islet-FasL组(n=21):移植经SA-FasL修饰的胰岛,(2)islet-SA组(n=21):移植经SA修饰的胰岛;(3)islet组(n=14):移植未经修饰的胰岛,作为空白对照.若受者尿糖阳性,并连续2d血糖≥250 mg/ml为移植排斥反应.尿糖阴性超过100d为移植物长期存活.结果 流式细胞术检测FasL蛋白在脾细胞的平均表达率为(94.49±4.27)%,免疫荧光染色显示FasL蛋白呈红色,表达于脾细胞表面;FasL蛋白的表达干扰脾细胞表面分子CD3、CD4、CD8、CD80以及主要组织相容性复合物I类抗原的表达.SA-FasL组70%的受者移植心获得长期存活,SA组仅为25%,两组间的差异有统计学意义(P<0.05).胰岛移植术后30d,islet-FasL组67%的受者移植胰岛功能良好,29%的受者移植胰岛获得长期存活,与其他两组(均为0)比较,差异均有统计学意义(P<0.05).结论 ProtEx~(TM)是简便、快速、安全和高效的外源蛋白修饰技术,过该技术将嵌合蛋白FasL直接表达于靶细胞,将对移植免疫应答发挥重要的调节作用.     

Preparation of an exogenous fibrin clot

Exogenous fibrin clot has been proposed to promote the healing of meniscal tears in areas of compromised vascularity. We present a method to reproducibly prepare a tightly wound exogenous fibrin clot using 5 to 10 mL of blood obtained by sterile venipuncture. The technique produces a clot of increased consistency and high fibrin content that is adaptable in preparation as a longer, thinner clot or a shorter, thicker clot. Clots prepared in this manner will hold suture or can be morselized for extrusion through a syringe.     

Reproductive outcomes following robotic-assisted laparoscopic myomectomy (RALM)

Uterine myomas are the most common type of benign tumor in women of reproductive age and are commonly associated with menorrhagia, dysmenorrhea, dyspareunia, and urinary symptoms. Uterine fibroids have been also linked to infertility and pregnancy loss. Women wishing to preserve or restore their fertility are best treated by myomectomy. Robotic-assisted laparoscopic myomectomy is one of the latest technological applications of minimally invasive surgery. Limited data exist regarding the feasibility of robotic-assisted laparoscopic myomectomy (RALM) in terms of pregnancy and surgical outcomes, and more studies are needed. The goal of this study is to assess reproductive outcomes following RALM in a private practice setting. The study was performed in the form of a retrospective chart review. Female patients 22–44 years old diagnosed with intramural myoma were eligible for inclusion. Presence of a myoma was a necessary but not necessarily the presenting symptom. All patients underwent RALM between January 2006 and May 2009 under the care of one surgeon at two clinical sites. Patients were selected postsurgically via chart review based on inclusion parameters outlined above. Median values for pregnancy rate, number of myomas, diameter of largest myoma, surgery duration, and blood loss were calculated and used for subsequent statistical analysis. Clinically useful markers for pregnancy outcomes evaluation following RALM were identified. Thirty patients were enrolled, of whom 16 were interested in conception due to infertility. A pregnancy rate of 75 % was recorded. Among those who conceived, eight patients (67 %) reported natural conception within 6 months of unprotected intercourse, while four patients (33 %) utilized assisted reproductive technologies to conceive. One patient (8 %) miscarried. Two patients (17 %) experienced premature delivery, at 28 and 32 weeks, respectively. All deliveries were performed via Cesarean section. No surgical complications were reported following RALM. There were no cases of scar dehiscence or rupture. The median number of myomas in those who delivered was estimated at 1.0 compared with 3.5 in those unable to conceive (p < 0.05). In addition, median age was 34 compared with 42.5 years, respectively (p < 0.05). This retrospective study assessed pregnancy outcomes following RALM. Our pregnancy rate of 75 % combined with a low incidence of complications contributes to the limited pool of data available on this topic, and supports the need for a multicenter trial to further evaluate effectiveness and safety of RALM in terms of pregnancy outcomes.     

Mice transgenic for an expanded CAG repeat in the Huntington's disease gene develop diabetes

The autosomal dominant neurological syndrome of Huntington's disease has been modeled in transgenic mice by the expression of a portion of the human huntingtin gene together with 140 CAG repeats (the R6/2 strain). The mice develop progressive chorea with onset at approximately 9 weeks of age and with death at approximately 13 weeks. Associated symptoms include weight loss and polyuria in the absence of eating or drinking deficits. We have found that these mice have insulin-responsive diabetes. Fasting glucose was 211 + 19 mg/dl in R6/2 mice compared with 93 + 5 mg/dl in C57/B6 controls (n = 12, both groups; P < 0.01). Administration of insulin intraperitoneally led to a reduction in blood glucose. At 12.5 weeks, animals were killed and pancreas weighed and analyzed for insulin and glucagon. Pancreatic mass in R6/2 mice was the same as controls, and islets appeared normal in morphology without lymphocytic infiltration. Immunohistochemical staining showed dramatic reductions in glucagon in the alpha-cells and in insulin in the beta-cells. Direct tissue assays showed glucagon and insulin content were reduced to only 10 and 15% of controls, respectively. Diabetes has been reported as being more common in Huntington's disease and other triplet repeat disorders. The R6/2 mouse should prove useful for elucidating the mechanism of diabetes in these genetic diseases.     

Elevated serum triiodothyronine (t3) in Ashkenazi Jewish prostate cancer patients carrying the I1307k allele of the APC (adenopolyposis coli) gene

PURPOSE: The risk of developing any cancer in carriers of the I1307K mutation of the adenopolyposis coli (APC) gene is significantly increased (odds ratio 1.5, P = 0.01). One of the cancers associated with the I1307K mutation is prostate cancer (odds ratio 2.0, P = 0.14). Also, there is an association of APC mutations with thyroid cancer. In this study, we measured triiodothyronine (t3) levels in Ashkenazi Jewish prostate cancer patients, with and without the I1307K mutation of the APC gene. MATERIALS AND METHODS: Participants in our study were found through urology and radiation oncology clinics in 1999 and 2000. All eligible patients were asked to take part. All patients had been initially diagnosed on the basis of rising PSA or abnormal physical examination. Histological confirmation of diagnosis was obtained for all subjects. Ethnic background was confirmed for all subjects by self-report or interview. The I1307K allele of the APC gene was detected by amplification of DNA isolated from peripheral blood according to standard polymerase chain reaction (PCR) and dot blot procedures. Serum t3 level was determined by fluorescent immunoassay with a standard, commercially available instrument. RESULTS: We studied 77 patients. The youngest patient was 46, the oldest 88, average age 67 +/- 7.2 (mean +/- SD). Eleven males carrying the APCI 1307K allele had significantly higher serum t3 levels than 66 males carrying the wild type allele. There were no homozygotes for the I1307K allele. None of the males had a t3 level that was above the normal range for our laboratory (137 ng/dl). CONCLUSIONS: Our findings of increased serum t3 level with the APC I1307K allele in prostate cancer patients is not surprising, given the mitogenic potential of t3. Further studies may clarify whether t3 elevation is the mechanism whereby APC gene mutations increase the risk of prostate cancer, or whether other pathophysiologic abnormalities are involved.     

p53病毒增强型质粒在血管平滑肌细胞中的表达

目的研究腺相关病毒增强型质粒载体介导的P~（53）基因在血管平滑肌细胞中的表达。方法构建带有野生型P~（53）基因的腺相关病毒增强型质粒表达载体，通过阳离子脂质体介导，转染血管平滑肌细胞。PCR检测外源基因整合，RT－PCR分析外源基因mRNA的表达，免疫组织化学检测基因表达产物。结果P~（53）基因转染血管子滑叽细胞表达相应的mRNA和蛋白质。结论血管平滑肌细胞可作为心血管疾病基因治疗的靶细胞．所构建的P~（53）腺相关病毒增强型质粒可作为基因治疗的载体。     

携带Fas基因重组腺病毒治疗瘢痕疙瘩的体内实验研究

目的 研究携带人Fas基因的两种重组腺病毒对瘢痕疙瘩的体内治疗效果。方法 构建瘢痕疙瘩裸鼠模型,应用携带Fas基因的常规腺病毒Ad—Fas（T）和细菌内重组腺病毒Ad—Fas（B）注射及Fas单克隆抗体（FasMcab）辅助对植入裸鼠皮下的瘢痕疙瘩组织进行治疗。通过大体观察、常规病理及电镜观察检测瘢痕疙瘩组织的变化。结果 单纯使用腺病毒注射的瘢痕疙瘩组织块体积仅轻度缩小。前期注射Ad—Fas（B）或Ad—Fas（T）后,应用FasMcab作为后续治疗,瘢痕疙瘩组织块均明显缩小,HE染色证实瘢痕疙瘩组织结构遭到破坏,电镜观察发现细胞凋亡证据。结论 重组腺病毒Ad—Fas（B）及Ad—Fas（T）的瘢痕疙瘩基因治疗效果令人满意。为瘢痕疙瘩的治疗提供了新途径。     

Increased collagenolytic activity in severed and sutured tendons following topical application of exogenous collagen in chickens

To verify the role of collagenase in reduction of peritendinous adhesion by topical application of exogenous collagen, the flexor tendons of 30 chickens were severed and sutured. Exogenous, native enriched collagen solution (ECS) was introduced in the tendon sheath via a polyethylene catheter. The effect of ECS on collagenolytic activity in the healing tendon was assessed 1, 2, and 3 weeks later both by determining the relative amounts of dialyzable protein and hydroxyproline and by using the collagen film collagenase assay. The results obtained indicated a significant increase in both dialyzable hydroxyproline level and collagenolytic activity in the ECS-treated tendons as compared with the untreated controls. It is suggested that the effect of the topically applied exogenous collagen on increasing the collagenolytic activity may be directly related to previously observed increased gliding capacity of the tendons in the same experimental model.