發(fā)布時間：2018-06-28 15:12

  本文選題：丹參 + 人脂肪干細(xì)胞　； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:將丹參與人脂肪來源干細(xì)胞聯(lián)合,通過動物實驗,探討二者綜合作用對自體脂肪顆粒植入裸鼠皮下后成活率的影響,進(jìn)而指導(dǎo)臨床應(yīng)用。方法:體外分離、培養(yǎng)、鑒定hADSCs:脂肪取自接受吸脂手術(shù)的1名25歲健康女性的腹部,兩次吸脂手術(shù)皆是求美者自愿捐贈。取出的脂肪置于無菌離心管中適量的,加入適量磷酸鹽緩沖鹽水(PBS),運(yùn)用離心法將脂肪顆粒純化,去除位于離心管上層的油脂、及位于離心管下層的雜質(zhì)后,剩余的脂肪顆粒用于hADSCs的制備,第二次抽取的脂肪顆粒同樣的方法處理后將脂肪顆粒置于無菌射器中為脂肪顆粒移植備用。選取24只8周齡健康雌性裸鼠,體重(23±3)g,隨機(jī)分為四組(n=6),A組:脂肪顆粒0.5ml;B組:脂肪顆粒0.5ml+hADSCs;C組:丹參注射液(0.5g·kg~(-1)·d~(-1))[1]+脂肪顆粒0.5ml;D組:丹參注射液(0.5g·kg~(-1)·d~(-1))+脂肪顆粒0.5ml+hADSCs。干細(xì)胞與顆粒脂肪比例為:6×105:0.5ml。A、B兩組每天腹腔注射與C、D組的丹參注射液等體積的生理鹽水。將脂肪顆粒按規(guī)定移植入各組裸鼠背部兩側(cè)對稱的皮下。移植術(shù)后15天每組隨機(jī)選取3只裸鼠處死后取材。取材后,首先對標(biāo)本進(jìn)行大體觀察,觀察組織的體積大小、包膜情況,包膜表面的毛細(xì)血管形態(tài)特征。然后將標(biāo)本放入10%甲醛中固定,常規(guī)石蠟包埋,切片,將每塊標(biāo)本做5張切片,1張切片用于HE染色,剩余4張切片為免疫組織化學(xué)染色備用,用于觀察并計數(shù)微血管個數(shù)。脂肪顆粒移植術(shù)后第30天處死各組剩余的裸鼠,將取出的標(biāo)本進(jìn)行體積測量。然后將標(biāo)本用10%甲醛中固定,常規(guī)石蠟包埋,切片,每塊標(biāo)本取一張切片做HE染色。結(jié)果:1人脂肪干細(xì)胞分離、培養(yǎng)及鑒定:細(xì)胞提取物由DMEM培養(yǎng)液重懸后,將細(xì)胞懸液接種于75cm2的無菌細(xì)胞培養(yǎng)瓶中,培養(yǎng)7-9天后培養(yǎng)瓶中可觀察到有大量梭形細(xì)胞及少量的多角形細(xì)胞,多角形細(xì)胞幾天后會逐漸變形為梭形細(xì)胞,14天左右可觀察到梭形細(xì)胞呈旋渦狀或束狀生長。由于原代培養(yǎng)脂肪組織中混有少量血管等其他的組織,因此培養(yǎng)瓶中會培養(yǎng)出個別紅細(xì)胞和其他雜質(zhì)細(xì)胞,而隨著脂肪干細(xì)胞的傳代培養(yǎng),脂肪干細(xì)胞越來越純。當(dāng)培養(yǎng)達(dá)13~(-1)4天時,原代細(xì)胞能達(dá)到80%融合,即可進(jìn)行傳代。傳代后細(xì)胞增長速度加快,每4-5天即可傳1代。2流式細(xì)胞術(shù)檢測:檢測結(jié)果表明CD29、CD44、CD90的表達(dá)率均在92%以上;CD31、CD45、CD146陽性率均小于6%,是幾乎不表達(dá)的,證明該實驗中所培養(yǎng)的細(xì)胞確實是hADSCs,且h ADSCs屬于間充質(zhì)干細(xì)胞的一種。3移植物組織學(xué)肉眼觀察:移植術(shù)后第15天,取出的移植體均可見完整的包膜形成,可見新生毛細(xì)血管走形在包膜表面,幾組結(jié)果對比,新生的毛細(xì)血管最為豐富的是D組移植物,新生毛細(xì)血管最少的是A組,B,C兩組的毛細(xì)血管數(shù)量介于A、D兩組之間;移植術(shù)后第30天,移植物的體積D組最大,A組的體積最小,B、C兩組的體積大小介于A、D兩組體積大小之間。4免疫組化檢測移植脂肪的微血管數(shù)量:對術(shù)后15天各組脂肪移植體免疫組化切片血管密度值采用LSD-t法進(jìn)行兩兩比較發(fā)現(xiàn),A組與B組,A組與C組,A組與D組,B組與D組,C組與D組之間的差異具有統(tǒng)計學(xué)意義(P0.05),而B組與C組之間的差異無統(tǒng)計學(xué)意義(P0.05),可以認(rèn)為D組的血管密度值最高,A組的血管密度值最低,B組和C組的血管密度值介于A組與D組之間。5丹參聯(lián)合hADSCs對移植顆粒脂肪體積的影響:術(shù)后30天各組脂肪移植體體積值采用LSD-t法進(jìn)行兩兩比較發(fā)現(xiàn),A組與B組,A組與C組,A組與D組,B組與D組,C組與D組之間的差異具有統(tǒng)計學(xué)意義(P0.05),而B組與C組之間的差異無統(tǒng)計學(xué)意義(P0.05),可以認(rèn)為D組的脂肪移植體體積值最高,A組的脂肪移植體體積值最低,B組和C組的脂肪移植體體積值介于A組與D組之間。結(jié)論:丹參與hADSCs的綜合作用,比各自單獨(dú)運(yùn)用更能有效地促進(jìn)脂肪顆粒移植體早期血管系統(tǒng)的重建,更有效地提高脂肪顆粒成活率。
[Abstract]:Objective: to combine Salvia miltiorrhiza with human fat derived stem cells and through animal experiments, the effects of the two synthetic effects on the survival rate of autologous fat particles implanted in nude mice were investigated, and then the clinical application was guided. Methods: in vitro isolation, culture, and identification of the abdominal and two liposuction hands of the hADSCs: fat from 1 25 year old women who received liposuction. The fat is placed in the aseptic centrifuge tube. The fat particles are added to the aseptic centrifuge tube, and a proper amount of phosphate buffer salt water (PBS) is added. The fat particles are purified by centrifugation to remove the fat in the upper layer of the centrifuge tube, and after the impurities located in the lower layer of the centrifuge tube, the remaining fat particles are used for the preparation of hADSCs and the second extraction of fat. 24 8 weeks old healthy female nude mice were selected and 24 8 weeks old healthy female nude mice were selected. The body weight (23 + 3) g was randomly divided into four groups (n=6), group A: fat granule 0.5ml; B group: fat granule 0.5ml+hADSCs; C group: Dan Shen Injection (0.5g. Kg~ (-1). D~ (-1)) The ratio of 0.5g / kg~ (-1) / d~ (-1)) + fat particles 0.5ml+hADSCs. stem cells and granular fat was 6 * 105:0.5ml.A, B two was injected into the abdominal cavity every day with C and Salvia miltiorrhiza injection in the D group. The fat particles were transplanted into the subcutaneous symmetry on both sides of the back of the nude mice in each group. 3 nude mice in each group were randomly selected for 15 days after the transplantation. After the rats were sacrificed, the specimens were taken. First, the specimens were observed, the size of the tissue, the envelope, the morphology of the capillaries on the surface of the membrane were observed. Then the specimens were fixed in 10% formaldehyde, the paraffin was embedded and sliced, and 5 slices were made for each specimen, 1 slices were used for HE staining, and the remaining 4 slices were immunized. Thirtieth days after the transplantation of fat particles, the remaining nude mice were killed and the samples were measured. Then the specimens were fixed in 10% formaldehyde, the paraffin was embedded and sliced, and each specimen was sliced for HE staining. Results: 1 human fat stem cells were separated, cultured and evaluated. The cell suspension was suspended by DMEM culture, and the cell suspension was inoculated into the sterile cell culture bottle of 75cm2. A large number of spindle cells and a small number of polygonal cells could be observed in the culture bottle for 7-9 days. The polygonal cells gradually became spindle cells after a few days, and the spindle shaped cells were observed to be vortexed or bundled at about 14 days. As the primary culture adipose tissue is mixed with a small amount of blood vessels and other tissues, a few red cells and other impurity cells will be cultivated in the culture bottle. With the generation of fat stem cells, the fat stem cells are becoming more and more pure. When the culture reaches 13~ (-1) 4 days, the primary cells can reach 80% fusion and can be passaged. The growth rate of the posterior cell was accelerated, and the 1 generation.2 flow cytometry was passed every 4-5 days. The results showed that the expression rate of CD29, CD44 and CD90 was above 92%; CD31, CD45, CD146 positive rates were less than 6%, which were almost not expressed. It proved that the cells cultured in this experiment were hADSCs, and H ADSCs belonged to a kind of.3 graft of mesenchymal stem cells. Histology naked eye observation: fifteenth days after transplantation, all the transplanted bodies were found to form a complete capsule, and the new capillaries were visible on the surface of the capsule. The most abundant new capillaries were group D grafts and the new capillaries were the A group, and the number of capillaries in group B and C two were between A and D two groups. Thirtieth days after transplantation, the size of the D group was the largest, the volume of the A group was the smallest, the volume of the group of B, the two group was between A, and the volume size of the D two groups was measured by.4 immunohistochemistry. The number of the blood vessels of the transplanted fat was detected by the LSD-t method at 15 days after the operation, and the A group and B group, A. The difference between group A and group C, group B and group D, group B and D, C group and D group was statistically significant (P0.05), but the difference between group B and C group was not statistically significant (P0.05). It was considered that the density of the vessel in the D group was the highest and the blood vessel density of the group was the lowest. The effect of the volume of grain fat: 30 days after the operation, the volume value of fat transplantation was compared with the LSD-t method for 22 comparison. The difference between group A and B, group A and C, group A and D, B and D, C group and D group had statistical significance (P0.05), but there was no significant difference between the group and the group. The volume value of the fat graft in group A is the lowest, the volume value of the fat graft in group B and group C is between A and D. Conclusion: the comprehensive effect of Salvia miltiorrhiza and hADSCs is more effective in promoting the reconstruction of the early vascular system of the fat granule transplantation, and more effectively improving the survival rate of fat particles.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R622.9

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