發(fā)布時(shí)間：2018-05-11 13:41

  本文選題：骨缺損 + 超聲微泡轉(zhuǎn)基因技術(shù)��； 參考：《中國(guó)修復(fù)重建外科雜志》2017年04期

【摘要】：目的探討超聲微泡轉(zhuǎn)基因技術(shù)介導(dǎo)增強(qiáng)型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)基因在兔骨缺損處轉(zhuǎn)染時(shí),不同超聲輻照時(shí)間對(duì)轉(zhuǎn)染效率及局部組織的影響。方法 3月齡雄性新西蘭大白兔30只,體質(zhì)量2.5~3.0 kg,制備右尺骨骨缺損模型,并隨機(jī)分為5組(n=6)。造模后第10天于骨缺損斷端間注射EGFP質(zhì)粒微泡混懸液(0.3 m L/kg)后,在超聲頻率1 MHz、超聲強(qiáng)度0.5 W/cm~2、占空比20%條件下,對(duì)骨缺損部位分別進(jìn)行1、2、3、4、5 min超聲輻照(分別為1、2、3、4、5 min組)。觀察動(dòng)物存活情況;轉(zhuǎn)染后1周取材,大體觀察骨缺損處軟組織形態(tài);熒光染色觀察基因表達(dá)情況;HE染色及透射電鏡觀察局部組織損傷情況。結(jié)果各組動(dòng)物均存活至實(shí)驗(yàn)完成。轉(zhuǎn)染后1周各組骨缺損處有軟組織生長(zhǎng),周?chē)∪饨M織部分內(nèi)陷填充于其間。熒光顯微鏡下觀察,各組兔骨缺損處均有綠色熒光表達(dá),其中2 min組表達(dá)最強(qiáng),1 min組表達(dá)最弱,其吸光度(A)值與其他各組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05);3、4、5 min組間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。HE染色及透射電鏡觀察示,各組骨缺損處局部均有不同程度組織損傷,損傷程度隨輻照時(shí)間的延長(zhǎng)而加重。結(jié)論超聲微泡轉(zhuǎn)基因技術(shù)介導(dǎo)EGFP質(zhì)粒在兔骨缺損部位轉(zhuǎn)染時(shí),其轉(zhuǎn)染效率和超聲輻照時(shí)間相關(guān)。當(dāng)超聲參數(shù)為1 MHz、0.5 W/cm~2、20%占空比時(shí),超聲輻照2 min可獲得最佳轉(zhuǎn)染效率及相對(duì)較輕的組織損傷。
[Abstract]:Objective to investigate the effect of different ultrasound irradiation time on the transfection efficiency and local tissue of enhanced green fluorescent protein (EGFP) gene in rabbit bone defect mediated by ultrasound microbubble technology. Methods Thirty 3-month-old male New Zealand white rabbits with a body mass of 2.5 and 3.0 kg were used to establish a model of right ulnar bone defect and were randomly divided into 5 groups. On the 10th day after the model was established, the EGFP plasmid microbubble suspension was injected between the broken ends of the bone defect (0.3 mL / kg). Under the condition of ultrasound frequency 1 MHz, ultrasound intensity 0.5 W / cm ~ (2) and duty ratio 20%, the site of the bone defect was irradiated with 1 ~ (2) ~ 2 ~ 3 ~ (3) ~ 3 ~ 4 ~ (5) min ultrasound irradiation (12 ~ 3X ~ 4 ~ 4 ~ 5 min group, respectively). The survival of animals was observed, the soft tissue morphology of bone defect was observed 1 week after transfection, the expression of gene was observed by fluorescence staining and the local tissue injury was observed by transmission electron microscope (TEM) and HE staining. Results all the animals survived until the experiment was completed. At 1 week after transfection, soft tissue growth was found in the bone defect of each group, and part of the surrounding muscle tissue was filled with soft tissue. The expression of green fluorescence was observed under fluorescence microscope in all groups, and the expression of green fluorescence was the weakest in the 2 min group, and the strongest in the 2 min group, and the weakest in the 1 min group. Compared with other groups, there was significant difference in absorbance A) value. There was no significant difference between the two groups (P 0.05). He staining and transmission electron microscope observation showed that there were different degrees of tissue injury in the bone defect in each group. The degree of injury increased with the prolongation of irradiation time. Conclusion the transfection efficiency of EGFP plasmid mediated by ultrasound microbubble gene transfer in rabbit bone defect is correlated with the time of ultrasound irradiation. The optimal transfection efficiency and relatively light tissue damage could be obtained by ultrasound irradiation for 2 min when the ultrasonic parameters were 0.5 W / cm ~ (2) ~ (2) duty cycle ratio of 1 MHz / cm ~ (2) C ~ (2 +).
【作者單位】： 四川大學(xué)華西醫(yī)院小兒外科;廣州市婦女兒童醫(yī)療中心小兒外科;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(81071475)~~
【分類(lèi)號(hào)】：R683

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本文編號(hào)：1874196