与基底细胞癌细胞凋亡相关的分子生物学研究--促凋亡分子Bad的克隆及其pEGFP-C3真核表达载体的构建

目的:克隆Bcl-2相关凋亡基因Bad并构建其真核表达载体,探讨其对肿瘤细胞凋亡的诱导作用。方法:采用RT-PCR的方法,扩增Bad基因全长片段。通过基因定向克隆,构建Bad基因的真核表达质粒载体。结果:经酶切鉴定、PCR及DNA序列测定鉴定,Bad基因表达质粒pEGFP-C3载体成功构建。结论:成功克隆Bcl-2相关促凋亡基因Bad并构建其真核表达载体pEGFP-C3-Bad,为进一步研究Bad在人肿瘤细胞系中的促凋亡作用奠定了实验基础。     

Biological activation of bone‐related biomaterials by recombinant adeno‐associated virus vector

Gene therapy is a promising clinical tool that is no longer limited as a method to supplement genetic deficits, but rather is considered reliable for delivering proteins to specific tissues or cells. Recombinant adeno‐associated virus (rAAV) vector is one of the most potent gene transfer vehicles. Many biomaterials have been used in reconstructive surgery, but their biological inactivity has limited their use. To overcome shortcomings of available bone‐related biomaterials, we investigated the combination of rAAV with biomaterials. Taking advantage of the method of lyophilizing rAAV onto biomaterials, we showed that an rAAV coating successfully induced β‐galactosidase protein expression by rat fibroblasts on hydroxyapatite, β‐tricalcium phosphate, and titanium alloy in vitro. β‐Galactosidase expression was detected for 8 weeks after implantation of rAAV‐coated hydroxyapatite into rat back muscles in vivo. A coating of bone morphogenetic protein‐2‐expressing rAAV induced significant de novo bone formation on hydroxyapatite in rat back muscles. Our study demonstrates that the combination of lyophilized rAAV and biomaterials presents a promising strategy for bone regenerative medicine. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

人源化绿色荧光蛋白基因真核表达载体的构建与表达检测

目的: 构建人源化绿色荧光蛋白(humanized greenfluorescent protein,hGFP)基因的真核表达栽体pcDNA3GFP,并观察其在MDCK细胞中的表达情况. 方法: 以Not I切取GFP后插入真核表达载体pcDNA3,以BamH I为鉴定方向,以Lipofect AMINE PLUSTM将pcDNA3GFP转染至培养的MDCK,经G418筛选GFP阳性克隆,于荧光显微镜下观察. 结果: 成功构建了含hGFP cDNA的真核表达载体 pcDNA3GFP,并成功转染MDCK细胞,在荧光显微镜下阳性克隆呈绿色. 结论: GFP基因可在MDCK细胞中成功表达,并发出绿色荧光,是一良好的报告基因和筛选标记.     

Recoverability of renal functions after relief of partial ureteric obstruction of solitary kidney: impact of ferulic acid

What's known on the subject? and What does the study add? It is known that the kidney damage continues even after release of ureteric obstruction. This study found that giving ferulic acid, antioxidant, after release of ureteric obstruction enhanced the recovery of kidney functions in solitary kidney.

OBJECTIVE

• ? To evaluate the effect of ferulic acid (FA) on the recovery of renal function and renal damage after relief of partial ureteric obstruction (PUO) of a solitary kidney.

METHODS

• ? Male mongrel dogs (n= 32) were classified into three groups: sham (eight), control (12) and study (12).
• ? A right nephrectomy was carried out and dogs in the study and control groups were subjected to 4 weeks of PUO.
• ? Serum creatinine, creatinine clearance (CrCl) and renographic clearance (RC) were measured at baseline, after 4 weeks of obstruction and 8 weeks after relief of obstruction.
• ? Markers of lipid peroxidation (malondialdehyde [MDA]), superoxide dismutase (SOD), and reduced glutathione (GSH), and immunostaining of markers of apoptosis (caspase 3 and Bcl2), cell proliferation (Ki67) and interstitial fibrosis in the kidney were evaluated at the end of experiment.

RESULTS

• ? Ferulic acid enhanced the recovery of serum creatinine, CrCl and RC by an extra 22%, 26% and 33.7%, respectively, of the basal values at 8 weeks, after relief of 4 weeks' obstruction.
• ? In addition, FA caused a significant decrease in MDA and a significant increase in GSH and SOD.
• ? Ferulic acid also significantly reduced the interstitial fibrosis, and caspase 3 expression, and significantly increased the expression of Bcl2 and Ki67 in kidney tissues at 8 weeks after relief of the obstruction.

CONCLUSION

• ? Ferulic acid enhances the recoverability of renal function and minimizes the renal damage through reduction of oxidative stress, tubular apoptosis and the interstitial fibrosis in the solitary kidney after relief of PUO.

     

Effects of adeno‐associated virus‐2‐mediated human BMP‐7 gene transfection on the phenotype of nucleus pulposus cells

Bone morphogenetic protein‐7 (BMP‐7) was found to stimulate the synthesis of proteoglycans (PGs) and collagen type II. To increase the biological function of the nucleus pulposus (NP) cells, the Ad‐hBMP‐7 vector was also successfully constructed and transfected NP cells. However, the disadvantages of adenovirus limit the usefulness of the Ad‐hBMP7 vector for clinical application. The rAAV2 vector has empirical advantages, especially for clinical use, to transfer exogenous genes into cells. The purpose of this study was to first determine whether a rAAV2‐hBMP‐7 vector could be used to transfect canine NP cells and effect on the biological functions of canine NP cells. The canine NP cells transfected by the rAAV‐BMP7 were assessed semi‐quaiitatively for BMP‐7 expression with real‐time PCR and westernbloting. Aggrecan and collagens type I and II secreted by the NP cells were qualitatively assessed at 4, 7, and 14 days post‐transfection in the transfection and control groups. We found that rAAV2 can successfully transfer the hBMP‐7 gene into canine NP cells. NP cells transfected by the rAAV‐hBMP‐7 vector express hBMP‐7 for at least 14 days. At 7 and 14 days, the expressed hBMP‐7 promotes a remarkable and significant accumulation of both proteoglycans (42% and 77% higher than non‐transfected cells) (p<0.05) and collagen type II (63% and 94% higher than non‐transfected cells) (p<0.05). Thus, we could speculate that the rAAV‐based gene delivery technique promotes the expression of proteoglycans and collagen type II of nucleus pulposus cells. Moreover, this technique may be applicable for the future treatment of degenerative disc disease. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:838–845     

Anionic and cationic drug secretion in the isolated perfused rat kidney after neonatal surgical induction of ureteric obstruction

OBJECTIVE: To study the pathophysiological changes of renal tubular drug transport mechanisms in congenital renal obstruction, by developing a model for perfusing the isolated kidney (IPK) after neonatal surgical induction of partial ureteric obstruction in Hanover Wistar rats. MATERIAL AND METHODS: Moderately severe obstruction of the right kidney of male rats was created by burying a segment of the right ureter under the psoas fascia at 5-7 days after birth. Different fluorescent substrates for renal organic anion and cation drug transport systems were added to the IPK, and the concentration of these substances with time analysed in perfusate and urine. RESULTS: The reproducibility in all groups of the glomerular filtration rate (GFR) and drug excretion was remarkably good. GFR was significantly lower in obstructed kidneys than in unobstructed kidneys. 123Rhodamine, a marker for organic cation and P-glycoprotein transport, had a significantly lower maximum excretion rate in the obstructed than in unobstructed kidneys. Renal fractional clearance (123rhodamine clearance corrected for diminished GFR) was also significantly lower in obstructed kidneys. There was no significant difference in maximum excretion (absolute and corrected GFR) for Lucifer Yellow, a marker for sodium-dependent organic anion transport. The maximum excretion rate of calcein, a marker for sodium-independent organic anion transport, was significantly lower in the obstructed than in the unobstructed kidneys, but significantly higher after correcting for reduced GFR. CONCLUSION: The IPK is a good model for studying the effect of neonatal renal obstruction on tubular drug transport. These results show that organic anion and cation transport mechanisms are affected differently by obstruction.     

增强型绿色荧光蛋白基因转染小鼠骨髓间充质干细胞的研究

目的：以重组腺病毒（Ad）为载体,将增强型绿色荧光蛋白（EGFP）基因转染至小鼠骨髓间充质干细胞（mMSCs）中,为MSCs体内示踪提供实验基础。方法：用全骨髓细胞贴壁法分离纯化小鼠MSCs并体外扩增、鉴定,以重组绿色荧光蛋白基因的腺病毒（Ad-EGFP）转染,并用荧光显微镜和流式细胞仪检测转染率,CCK-8法检测转染细胞的增殖活性。结果：转染后10h MSCs开始表达荧光,6～8天达高峰,转染率可达92.3%,28天仍有表达;转染细胞的增殖活性和未转染细胞无统计学差异。结论：Ad-EGFP能高效、安全的转染mMSCs。     

Labeling of human insulin-like growth factor-I eukaryotic expression vector with green fluorescent protein

Articularcartilageinjuriesinadultsarecommon.Theintrinsicrepairisgenerallyminimalandcartilageinjuriescanfurtherdevelopintoosteoarthritis.Insulin likegrowthfactor I(IGF I)is regardedasoneofthemostimportantgrowthfactorsin cartilagedevelopmentandhomeostasis.Theadditionof IGF Itochondrocytesinvitroenhanceschondrocyte metabolism,maintainsadifferentiatedchondrocyte morphologyandpromotessynthesisofmajorcartilage matrixproteins,includingtype IIcollagenand proteoglycans.1,2IGF Iinlowconcentrationi…     

绿色荧光蛋白标记神经干细胞的体外研究

目的　构建携带绿色荧光蛋白 (GFP)基因的逆转录病毒载体pLNCX2 GFP ,用GFP对神经干细胞进行标记示踪。方法　应用基因克隆的方法 ,制备 pLNCX2 GFP ,借助阳离子脂质体转染包装细胞PA3 17,G418筛选阳性克隆 ,获取病毒上清 ;从胚胎大鼠脑中解剖分离和培养神经干细胞 ,用病毒上清感染大鼠胚胎神经干细胞。结果　经酶切电泳和DNA测序表明成功构建了重组GFP逆转录病毒 ,pLNCX2 GFP转染包装细胞后可以产生GFP逆转录病毒 ,病毒感染大鼠胚胎神经干细胞可以长期表达绿色荧光。结论　逆转录病毒能够快速、稳定、长期地将GFP基因转入神经干细胞 ,这种标记方法非常有利于神经干细胞移植后结构和功能的研究     

A porcine model of relief of 3 days unilateral ureteral obstruction: study on the damage and self-repairing capability of the kidney

Objective To explore the reversibility of early stage tubular interstitial injury as well as the timing of reparation through the pig relief of unilateral ureteral obstruction (R-UUO) model. Methods Eight three-month-old female Guangxi BA-MA mini pigs were selected for the construction of R-UUO models. Five time points were set which were UUO 0 day, UUO 3 days, R-UUO 7 days, R-UUO 14 days, and R-UUO 21 days. Renal function, histological structure, and protein expressions of α-smooth muscle actin (α-SMA), vimentin and E-cadherin were evaluated at different time points. Results After 3 days of UUO, compared with UUO 0 day, serum creatinine levels were increased obviously and the kidney tissues presented varying degrees of damage. The expressions of α-SMA and vimentin were increased and E-cadherin expression was decreased (P＜0.05). Following R-UUO after 3 days of UUO, compared to UUO, serum creatinine levels were significantly decreased. Renal pathological tissue damage was repaired. The expressions of α-SMA and vimentin were decreased and E-cadherin expression was increased (P＜0.05). Conclusions The pig R-UUO animal model may provide a good platform to study the kidney injury and repair. The tubular injury may be fully reversed and repaired when removing the pathogenic factors if the renal tubular injury was at an earlier stage.     

Minimally invasive surgical management of pelvic‐ureteric junction obstruction: update on the current status of robotic‐assisted pyeloplasty

BACKGROUND

Pelvi‐ureteric junction (PUJ) obstruction is characterized by a functionally significant impairment of urinary transport caused by intrinsic or extrinsic obstruction in the area where the ureter joins the renal pelvis. The majority of cases are congenital in origin; however, acquired conditions at the level of the ureteropelvic junction may also present with symptoms and signs of obstruction. Historically, open pyeloplasty and endoscopic techniques have been the main surgical options with the intent of complete excision or incision of the obstruction. The advent of laparoscopy and robotic‐assisted applications has allowed for minimally invasive reconstructive surgery that mirrors open surgical techniques.

AIMS

We review the current status of robotic‐assisted laparoscopic pyeloplasty and report on the result, continuing evolution, and potential role for this surgical procedure.

MATERIALS AND METHODS

A review of the recent literature encompassing laparoscopic and robotic‐assisted pyeloplasty was conducted with particular attention to operative techniques, surgical outcomes, and complication rates.

RESULTS

Laparoscopic and robotic‐assisted approaches are able to duplicate the open technique, and not surprisingly, are now being shown to be as efficacious as the gold standard open approach. The laparoscopic remains technically challenging due to the high proficiency level required for intracorporeal suturing, although added experience has resulted in shorter operative times. The advent of robotics has further expanded the breadth of this reconstructive procedure while preserving the benefits of decreased pain, shorter hospitalization, rapid convalescence, and an improved cosmetic result.

DISCUSSION

The introduction of robotics to the field of minimally invasive surgery facilitates this procedure and may allow for more widespread implementation by surgeons of varying skill levels. These benefits must be balanced against the increased costs of the robotic platform.

CONCLUSION

Clinical reports have demonstrated that robotic‐assisted pyeloplasty is a safe, feasible, and effective technique for treating ureteropelvic junction obstruction in short term studies. Additional studies with prolonged follow‐up will ultimately provide valuable information as to the long‐term efficacy of robotic‐assisted laparoscopic pyeloplasty.     

绿色荧光蛋白基因逆转录病毒载体转染人骨髓间充质干细胞及表达的研究

目的　绿色荧光蛋白 (EGFP)基因的逆转录病毒载体转染人骨髓间充质干细胞(MSCs)及其表达情况。方法　pEGFP与逆转录病毒载体经过酶切、连接等构建重组的EGFP 逆转录病毒载体 ,转染PA3 17细胞 ,用G418筛选出抗性克隆 ,获病毒上清 (滴度达到 8.5× 10 5cfu/ml)并感染人MSCs。流式细胞仪测定转染效率后加入成骨细胞分化培养夜 (地塞米松 (10 -7mol/L)、β 甘油磷酸钠 (10mmol/L)、维生素C(5 0mg/L ) ) ,2周后观察转染细胞的分化情况。 结果　 48h后细胞转染效率为 7% ,G418筛选 2周后约 97%细胞出现抗性克隆 ,表达维持 4周。基因转染细胞能够分化为成骨细胞。结论　重组EGFP逆转录病毒载体能转染MSCs ,且不影响细胞的功能。     

Expression of green fluorescent protein under beta-actin promoter in living spermatogenic cells of the mouse: stage-specific regulation by FSH

Spermatogenic cells from a mouse strain expressing enhanced green fluorescent protein (EGFP) under chicken beta-actin promoter were studied under living conditions to analyse stage- and cell-specific expression and hormonal regulation of the transgene. The isolated seminiferous tubules were examined by transillumination and the live cell squashes by phase contrast and fluorescence microscopy. FSH effects were measured in whole seminiferous tubules comparing stages I-VI, VII-VIII and IX-XII of the cycle. Beta-actin was highly expressed in spermatogonia, but almost no expression was found at early meiosis (leptotene spermatocytes). A gradual increase in translation of beta-actin was found during later stages of meiosis and early spermiogenesis, with a maximum in elongating spermatids. FSH increased the translation of beta-actin after 4 h and 24 h of incubation at stages I-VI, after 24 h at stages VII-VIII but not at stages IX-XII of the cycle. The results support the view that beta-actin plays a role in the nuclear elongation of spermatids and that its expression is regulated by FSH in a stage-specific fashion. Techniques used in this study give us new insight to study temporal and hormonal regulation of gene products in living spermatogenic cells.     

Apoptosis of circulating lymphocyte in rats with unilateral ureteral obstruction: role of angiotensin II

BACKGROUND: Unilateral ureteral obstruction (UUO) could induce increased renal angiotensin II (ANG II), which enhances apoptosis of renal tubular cells and renal tissue loss. Systemic ANG II is also increased in UUO. There are no data available about whether UUO can induce apoptosis of circulating lymphocytes or not. METHODS: UUO or sham-operated male Wistar rats (n = 8 in each group) were fed a drinking solution containing water, angiotensin II receptor type 1 antagonist (ARA; losartan, 500 mg/L) or angiotensin-converting enzyme inhibitor (ACEI; enalapril: 200 mg/L) for 1 day or 7 days. Blood samples were collected and circulating lymphocyte cells were separated. The apoptotic cells were detected by in situ terminal deoxynucleotidyl transferase (TdT assay)-mediated digoxigenin/antidigoxigenin conjugated fluorescein method and counted under a fluorescence microscope. The apoptotic index was calculated. RESULTS: UUO caused marked increases in the apoptotic index of circulating lymphocytes in UUO rats at both 1 day and 7 days when compared with the respective sham groups (P < 0.001). Neither ARA nor ACEI treatment had an effect on the apoptotic index values in the UUO rats at 1 day. In the UUO rats at 7 days, the apoptosis of circulating lymphocytes was markedly decreased from 29.2 +/- 2.7% to 11.9 +/- 2.7% (P < 0.01) in the ARA-treated rats and to 7.6 +/- 2.7% (P < 0.001) in the ACEI-treated rats. CONCLUSION: UUO, via stimulation of ANG II, could promptly enhance apoptosis of circulating lymphocytes. The apoptosis persisted throughout the 7 days of the study. Prolonged UUO would impair lymphocyte cell immunity and the host defense mechanism. Continuous treatment with either ARA or ACEI could abrogate ANG II-stimulated circulating lymphocyte apoptosis.     

Tracking of green fluorescent protein labeled Escherichia coli confirms bacterial translocation in blind loop rat

BACKGROUND: Previous investigators have documented small intestinal mucosal injury in blind loop rats. However, the definitive evidence of intestinal bacterial translocation in blind loop animals has been lacking. The purpose of this study was to confirm bacterial translocation in blind loop rats and to evaluate the preventive effect of glutamine on bacterial translocation caused by blind loops. MATERIALS AND METHODS: Escherichia coli TG1 labeled with green fluorescent protein was used to track bacterial translocation by gavage to rats. Six groups (n = 10) of rats were studied: unoperated control rats; rats with self-emptying blind loop; rats with self-filling blind loop; unoperated control rats treated with glutamine, 400 mg/d; rats with self-emptying blind loop treated with glutamine, 400 mg/d; rats with self-filling blind loop treated with glutamine, 400 mg/d. Representative tissue specimens of the mesenteric lymph nodes, liver, spleen, and kidney were aseptically harvested for bacteria culture. RESULTS: Bacteria were detected in extraintestinal organs of rats with self-emptying blind loop, self-filling blind loop, and self-filling blind loop treated with glutamine. By fluorescence microscope and XbaI restriction digestion analysis, we elucidated that the bacteria isolated from extraintestinal organs were the same bacteria we gavaged to the rats. CONCLUSION: We confirmed bacterial translocation in self-filling blind loop and self-emptying blind loop rats. In addition, we also showed that glutamine prevents bacterial translocation in self-emptying blind loop rats.