【摘要】：背景 腦血管疾病(Cerebral vascular disease, CVD)是指由各種病因使腦血管發(fā)生意外而導致腦功能缺損的一組疾病的總稱。腦血管疾病以其高發(fā)病率、高病死率和高致殘率嚴重影響著人們的生存及生活質(zhì)量,是多數(shù)國家的三大致死疾病之一。如何預防和治療腦血管疾病與及怎樣最大限度的降低腦血管疾病引起身體殘疾成為研究熱點,特別是當腦血管病發(fā)生后如何降低殘疾率就變的至關重要了。目前臨床上用于治療腦血管病后遺癥的方法眾多包括神經(jīng)營養(yǎng)因子、物理因子治療和運動功能鍛煉等方法,但仍然沒有一種方法可以很好地治療腦血管病引起的殘疾。隨著干細胞工程的推進,越來越多的學者注意到干細胞在疾病的治療當中具有重大的潛能,特別是運用干細胞修復受損組織,干細胞替代治療在組織損傷、遺傳缺陷、退行性等疾病中都有相關的研究,其中骨髓間充質(zhì)干細胞(bone marrow mesenchymal stem cells, BMSCs)是目前備受關注的一類組織干細胞。 間充質(zhì)干細胞是具有多方向分化潛能的成體干細胞,來源于中胚層間充質(zhì),主要存在于全身結締組織和器官間充質(zhì)中,以骨髓間充質(zhì)中含量最為豐富。在骨髓當中主要的干細胞為造血干細胞及其祖細胞,只有一部分為間充質(zhì)干細胞,BMSCs一方面是造血誘導微環(huán)境的重要成員,具有促進造血細胞增殖和分化的作用,參與支持和調(diào)控造血,幫助造血細胞粘附和歸巢；另一方面,在適當?shù)沫h(huán)境下,BMSCs可以分化為中胚層的細胞分化,甚至跨越胚層向外胚層及內(nèi)胚層來源的組織細胞分化。BMSCs容易獲得具有高增殖性,故可以體外大量增殖保證了充足的干細胞來源；BMSCs可以自體增殖然后移植,可避免免疫排斥問題同時在倫理道德上也沒有限制。正是由于BMSCs具有的這些特點,才使得BMSCs成為了干細胞的研究熱點。 對于臨床上的許多疾病都有相關的模型試驗應用BMSCs移植進行治療,包括心血管疾病、血液疾病、脊髓損傷、顱腦損傷和腦血管疾病等疾病。在腦血管疾病中腦卒中會給患者帶來嚴重的后遺癥,有許多研究都致力于如何運用BMSCs的移植治療腦血管病的后遺癥,相應的研究結果基本證實了BMSCs移植可以改善腦卒中的預后提高神經(jīng)功能。盡管許多學者都認可BMSCs移植治療腦血管病的存在有益作用,但在實際的運用中存在許多問題。BMSCs移植治療腦缺血疾病存在的問題包括移植途徑、移植細胞數(shù)、移植的時間窗、移植細胞的追蹤等,另一個存在的更重要的問題就是骨髓間充質(zhì)干細胞移植后存活率低的問題。組織缺血后,缺血區(qū)內(nèi)腦缺血缺氧造成腦組織周圍環(huán)境惡劣、微環(huán)境的改變；當BMSCs遷移入缺血區(qū)內(nèi)后由于生存環(huán)境的改變會導致BMSCs死亡造成細胞的存活率低,如果移植途徑不是定向移植再加上在循環(huán)過程中細胞的丟失,這樣能進入腦損傷部位的有活性的干細胞就會很少,這也是限制了BMSCs在臨床上推廣應用的一個難題。 物理因子治療腦血管病已經(jīng)成為了重要的臨床手段,并且得到了學者認可。然而目前臨床上應用的物理因子都有其優(yōu)勢和不足,急需科研工作者對潛在的物理因子進行研究,使更多的物理因子優(yōu)勢互補從而盡可能的降低腦血管病患者的致殘率。次聲是頻率在0.0001-20Hz之間的機械振動波,其廣泛存在于自然界。強度高于90dB的次聲已被研究證實對生物體具有損傷作用,強度低于90dB的次聲則在安全域內(nèi),我們把強度低于90dB的次聲稱為低聲壓次聲。對于低聲壓次聲具有的相關生物學效應卻鮮有研究,我們通過對低聲壓次聲的前期研究發(fā)現(xiàn)低聲壓次聲具有與高聲壓次聲相反的生理學作用,低聲壓次聲似乎對受損機體會存在治療作用。已有生理學研究結果表明,人體的各種器官都有一個固定的振動頻率,如頭部8-12Hz、胸部為4-6Hz、腹部為6-9Hz、盆腔為6Hz、心臟為5Hz。這些固有振動頻率都在次聲頻率范圍內(nèi),而次聲對機體作用的生物學機制就是能引起器官、組織的生物共振。對次聲的生物學效應的研究也從器官、組織深入到細胞,目前對細胞的研究主要是探討次聲對細胞的增殖、細胞周期、凋亡等生物學行為的影響。本實驗小組在前期的初步研究中,我們發(fā)現(xiàn)低聲壓次聲體外作用骨髓間充質(zhì)干細胞(BMSCs)60min后可明顯的促進細胞增殖抑制細胞凋亡,此結果預示著低聲壓次聲可以提高BMSCs的活性,為解決BMSCs移植后存活率低的問題提供了一種研究方法。 雖然我們的初步研究證明了低聲壓次聲能降低BMSCs的凋亡率提高其增殖活性,然而我們的前期研究在設計上不夠完善,為了更詳細地了解低聲壓次聲對BMSCs的影響及得到相關的干預參數(shù),本實驗的第一步完善低聲壓次聲對BMSCs的生物學效應影響的研究,接著深入探討低聲壓次聲對BMSCs生物學效應的影響與存活素(survivin)之間的關系,從而了解低聲壓次聲改變BMSCs生物學效應的相關分子學機制。 目的 深入研究低聲壓次聲對BMSCs的凋亡與增值的影響,完善干預參數(shù)真實性和可靠性,同時探討此影響與survivin之間的關系,進一步地了解低聲壓次聲對BMSCs作用,從而為低聲壓次聲與BMSCs的運用提供一些理論基礎。 研究方法 1.細胞獲取和培養(yǎng)：用頸椎脫位法處死SD大鼠,取大鼠后肢的股骨和脛骨去除兩端的骨骺,打開骨髓腔。用注射器吸取5mlDMEM/F12培養(yǎng)基反復沖洗骨髓腔,將沖洗后的骨髓懸液離心,倒掉上清液,用含10%胎牛血清的DMEM/F12培養(yǎng)基10ml重懸細胞,采用全骨髓貼壁培養(yǎng)法獲取細胞。 2. BMSCs的傳代和純化：利用差異貼壁培養(yǎng)法純化細胞,細胞首次按1：3傳代,第二次與第三次傳代按1：2傳代方法。每次傳代后做好標記P1、P2、P3及日期等的標記,放入37℃、5%C02孵箱中培養(yǎng)。本實驗使用的細胞為第三代處于對數(shù)生長期的細胞。 3.試驗處理及指標檢測： (1)臺盼藍染色：為了保證用于實驗的細胞有較高的活性降低實驗誤差,在細胞進行次聲處理前對各組使用臺盼藍染色方法進行細胞活力檢測。 (2)增殖與凋亡檢測：取P3代細胞,分試驗組和對照組,其中試驗組用次聲分別干預in、90min、120min,對照組暴露空氣中相同時間。處理結束后檢測細胞的增殖與凋亡,重復三次。 (3) survivin定性及定量檢測：取P3代細胞,分試驗組和對照組,試驗組用次聲干預60min,結束后放入細胞孵箱中培養(yǎng)72h,然后免疫熒光及qRT-PCR檢測細胞survivin的表達含量。 統(tǒng)計方法 采用spss13.0統(tǒng)計軟件對數(shù)據(jù)進行分析,各組數(shù)據(jù)以(x±s)表示。OD值及凋亡率的檢測采用方差分析,survivin表達量使用獨立樣本t檢驗分析,以P0.05作為統(tǒng)計學差異檢驗標準。 結果 1.兩組培養(yǎng)的P3代細胞存活率都在95%以上,說明細胞活力尚好,可以進行下一步實驗研究。細胞活性率=活細胞總數(shù)／(活細胞總數(shù)+死細胞總數(shù))×100% 2.實驗組的OD值：60min (1.560±0.075),90min (1.160±0.096),120min (0.930±0.069);對照組OD值：60min (1.239±0.025),90min (1.090±0.110),120min (0.926±0.027)。結果顯示實驗組60min的OD值顯著大于對照組60min,有統(tǒng)計學差異P0.01,90min和120min在實驗組與對照組中無統(tǒng)計學差異,實驗組中60min、90min與120min三組存在統(tǒng)計學差異,60minOD值最大,結果有統(tǒng)計學差異。結果證明低聲壓次聲干預BMSCs60min可使細胞存在最大的增殖活性。 3.實驗組的凋亡率：60min(10.70±1.47),90min(21.33±1.93),120min (22.87±1.45)；對照組凋亡率值：60min(18.20±1.12),90min(22.93±1.79),120min (23.47±2.50)。實驗組的凋亡率在60min顯著低于對照組60min的凋亡率(P0.01),90min和120min在實驗組與對照組中凋亡率都較高,在實驗組60min、90min與120min三組中,60min組凋亡率最低,結果有統(tǒng)計學差異。結果證明次聲干預BMSCs60min可顯著降低細胞的凋亡率。 4.通過觀察低聲壓次聲對BMSCs增殖與凋亡的影響,我們發(fā)現(xiàn)低聲壓次聲在60min不僅可以提高BMSCs增殖活性還可以降低細胞的凋亡率,基于此結果我們認為60min為低聲壓次聲干預BMSCs的一個理想的時間參數(shù),故檢測survivin的研究只觀察低聲壓次聲干預BMSCs60min。免疫組化結果顯示實驗組的BMSCs表達的survivin熒光強度顯著強于對照組,實驗組survivin的陽性率細胞計數(shù)(59.9±6.1),對照組survivin的陽性率細胞計數(shù)(24.3±5.8),兩者的差異有統(tǒng)計學意義(P0.05)。為了更準確地知道次聲對BMSCs表達survivin的影響,我們采用qRT-PCR定量檢測survivin的含量；實驗組survivin的mRNA的相對含量(1.318±0.051),對照組survivin的mRNA的相對含量(0.966±0.034),有統(tǒng)計學差異P0.01。結果證明低聲壓次聲可顯著地提高BMSCs表達survivin。 結論 1.低聲壓次聲可以促進BMSCs的增殖,降低細胞的凋亡,通過綜合分析各個干預時間點的結果,我們發(fā)現(xiàn)低聲壓次聲在60min對BMSCs的生物學作用最大,據(jù)此我們可以推斷60min為最理想的時間干預參數(shù)。 2.低聲壓次聲可以在促進BMSCs增殖抑制細胞凋亡的同時存活素的表達水平也明顯提高,據(jù)此我們初步推斷低聲壓次聲促進BMSCs增殖、抑制其凋亡的可能機制為次聲提高了BMSCs分泌survivin的能力,然而是否存在直接的相關關系還需進一步的研究。
[Abstract]:background
Cerebral vascular disease (CVD) refers to a group of diseases caused by various causes of cerebrovascular accidents leading to brain function deficits. Cerebral vascular disease with its high incidence, high mortality and high disability rate seriously affects people's survival and quality of life, is one of the three major death diseases in most countries. Prevention and treatment of cerebrovascular diseases and how to minimize the physical disability caused by cerebrovascular diseases have become a research hotspot, especially when cerebrovascular diseases occur, how to reduce the rate of disability becomes crucial. With the development of stem cell engineering, more and more scholars have noticed that stem cells have great potential in the treatment of diseases, especially in the use of stem cells to repair damaged tissues, stem cell replacement therapy in tissues. Bone marrow mesenchymal stem cells (BMSCs) are a kind of tissue stem cells which have attracted much attention.
Mesenchymal stem cells (MSCs) are adult stem cells with multi-directional differentiation potential. They are derived from mesenchymal mesenchyme and mainly exist in connective tissue and organ mesenchyme of the whole body. On the one hand, BMSCs are important members of the hematopoietic induction microenvironment, which can promote the proliferation and differentiation of hematopoietic cells, participate in supporting and regulating hematopoiesis, help hematopoietic cells adhere and homing; on the other hand, BMSCs can differentiate into mesodermal cells under appropriate conditions, even transcend the embryonic layer to tissues derived from the ectoderm and endoderm. BMSCs are easy to obtain and have high proliferative ability, so they can proliferate in vitro to ensure adequate stem cell sources; BMSCs can proliferate and transplant themselves, which can avoid the problem of immune rejection and has no ethical limitations. It is precisely because of these characteristics of BMSCs that BMSCs have become a hot topic in stem cell research. Point.
BMSCs transplantation is used to treat many clinical diseases, including cardiovascular diseases, blood diseases, spinal cord injury, brain injury and cerebrovascular diseases. Stroke can cause serious sequelae in patients with cerebrovascular diseases. Many studies have focused on how to use BMSCs transplantation treatment. BMSCs transplantation can improve the prognosis of cerebral apoplexy and improve neurological function. Although many scholars have recognized the beneficial effect of BMSCs transplantation in the treatment of cerebrovascular diseases, there are many problems in the practical application. Another more important problem is the low survival rate of bone marrow mesenchymal stem cells after transplantation. The survival rate of BMSCs is low because of the change of living environment. If the transplantation route is not directional transplantation and the loss of cells in the circulation process, there will be few active stem cells which can enter the brain injury site. This also restricts the clinical application of BMSCs.
Physical factors have become an important clinical means for the treatment of cerebrovascular diseases and have been recognized by scholars. However, the physical factors used in clinical practice have their advantages and disadvantages. It is urgent for researchers to study the potential physical factors so as to make more physical factors complement each other so as to reduce the incidence of cerebrovascular diseases as much as possible. Infrasound is a mechanical vibration wave with frequencies ranging from 0.0001 Hz to 20 Hz. It exists widely in nature. Infrasound with intensity higher than 90 dB has been proved to be harmful to organisms. Infrasound with intensity lower than 90 dB is in a safe range. Infrasound with intensity lower than 90 dB is called low pressure infrasound. Previous studies of low-pressure infrasound have found that low-pressure infrasound has opposite physiological effects to high-pressure infrasound, and low-pressure infrasound seems to have therapeutic effects on the injured organism. For example, the head is 8-12 Hz, the chest is 4-6 Hz, the abdomen is 6-9 Hz, the pelvic cavity is 6 Hz, the heart is 5 Hz. These natural vibration frequencies are all in the range of infrasound frequency, and the biological mechanism of infrasound on the body is to cause the biological resonance of organs and tissues. In our previous preliminary study, we found that low-pressure infrasound could significantly promote the proliferation of bone marrow mesenchymal stem cells (BMSCs) and inhibit cell apoptosis after 60 minutes in vitro. This result indicates that low-pressure infrasound can promote cell proliferation and inhibit cell apoptosis. In order to improve the activity of BMSCs, it provides a research method to solve the problem of low survival rate after BMSCs transplantation.
Although our preliminary studies have proved that low-pressure infrasound can reduce the apoptosis rate of BMSCs and increase their proliferation activity, our previous studies are not perfect in design. In order to understand the effect of low-pressure infrasound on BMSCs in more detail and obtain the relevant intervention parameters, the first step of this experiment is to improve the biological effects of low-pressure infrasound on BMSCs. Secondly, the relationship between the effect of low-pressure infrasound on the biological effect of BMSCs and Survivin was discussed in detail, so as to understand the molecular mechanism of low-pressure infrasound on the biological effect of BMSCs.
objective
To study the effect of low-pressure infrasound on apoptosis and proliferation of BMSCs, to improve the authenticity and reliability of intervention parameters, and to explore the relationship between this effect and survivin, to further understand the effect of low-pressure infrasound on BMSCs, so as to provide some theoretical basis for the application of low-pressure infrasound and BMSCs.
research method
1. Cell acquisition and culture: SD rats were killed by cervical vertebral dislocation, femur and tibia of hind limbs were removed, and bone marrow cavity was opened. Cells were obtained by whole bone marrow adherent culture.
2. The passage and purification of BMSCs: The cells were purified by differential adherence culture. The first passage was 1:3, and the second and third passages were 1:2. After each passage, the cells were labeled with P1, P2, P3 and date. The cells were incubated in incubators at 37 C and 5% C02. The cells used in this experiment were fine in logarithmic phase of the third generation. Cell.
3. test processing and target detection:
(1) Trypan blue staining: In order to ensure that the cells used in the experiment have high activity and reduce the experimental error, the cell viability was detected by trypan blue staining before infrasound treatment.
(2) Proliferation and apoptosis: P3 cells were divided into experimental group and control group. Infrasound was used in the experimental group for in, 90 and 120 minutes respectively. The control group was exposed to air for the same time.
(3) Qualitative and quantitative detection of survivin: P3 cells were divided into experimental group and control group. The experimental group was treated with infrasound for 60 minutes, then cultured in incubator for 72 hours. The expression of survivin was detected by immunofluorescence and qRT-PCR.
statistical method
The data were analyzed by SPSS 13.0 statistical software. The OD value and apoptosis rate were detected by variance analysis. The expression of survivin was analyzed by independent sample t test, and P 0.05 was used as the standard of statistical difference test.
Result
1. The survival rates of P3 cells cultured in both groups were above 95%, indicating that the cell viability was still good and could be further studied.
2. OD value of experimental group: 60 min (1.560.075), 90 min (1.160.096), 120 min (0.930.069); control group: 60 min (1.239.025), 90 min (1.090.110), 120 min (0.926.027). The results showed that the OD value of experimental group in 60 min was significantly higher than that of control group in 60 min (P 0.01, 90 min and 120 min). The results showed that the maximum proliferative activity of BMSCs could be obtained after 60 min, 90 min and 120 min of infrasound treatment.
3. The apoptotic rate of the experimental group was 60 min (10.70.47), 90 min (21.33.93), 120 min (22.87.45); the apoptotic rate of the control group was 60 min (18.20.12), 90 min (22.93.79), 120 min (23.47.50). The apoptotic rate of the experimental group was significantly lower than that of the control group in 60 min (P 0.01), 90 min and 120 min. The apoptotic rate was the lowest in 60 min, 90 min and 120 min groups. The results showed that infrasound could significantly reduce the apoptotic rate of BMSCs after 60 min.
4. By observing the effect of low-pressure infrasound on proliferation and apoptosis of BMSCs, we found that low-pressure infrasound could not only increase the proliferation activity of BMSCs, but also reduce the apoptosis rate of BMSCs at 60 minutes. Based on this result, we think that 60 minutes is an ideal time parameter for low-pressure infrasound to interfere with BMSCs, so the study of detecting survivin only observed low-pressure infrasound. The results of immunohistochemistry showed that the survivin fluorescence intensity of BMSCs in the experimental group was significantly stronger than that in the control group. The positive rate of Survivin in the experimental group was 59.9 (+ 6.1) and the positive rate of Survivin in the control group was 24.3 (+ 5.8). The difference was statistically significant (P 0.05). The relative content of Survivin mRNA in the experimental group (1.318.051) and the relative content of Survivin mRNA in the control group (0.966.034) were significantly higher than those in the control group (P 0.01).
conclusion
1. Low-pressure infrasound can promote the proliferation of BMSCs and reduce the apoptosis of cells. Through comprehensive analysis of the results of each intervention time point, we found that low-pressure infrasound has the greatest biological effect on BMSCs in 60 minutes, and we can infer that 60 minutes is the best time intervention parameter.
2. Low-pressure infrasound can promote the proliferation of BMSCs and inhibit the apoptosis of BMSCs at the same time, the expression of survivin is also significantly increased. Therefore, we preliminarily concluded that low-pressure infrasound can promote the proliferation of BMSCs and inhibit the apoptosis of BMSCs. The possible mechanism is that infrasound can improve the ability of BMSCs to secrete survivin. However, whether there is a direct correlation needs further study. Research.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R743

【參考文獻】

相關期刊論文 前10條

1 袁華,陳景藻,李玲,饒志仁,邱建勇;次聲作用后大鼠腦中熱休克蛋白70的表達和分布[J];第四軍醫(yī)大學學報;1998年06期

2 費舟,章翔,王曉峰,劉先珍,陳景藻,賈克勇;次聲作用后大鼠血腦屏障的改變及意義[J];第四軍醫(yī)大學學報;1999年08期

3 葉琳,龔書明,黃曉峰,王楓,劉靜,陳景藻;次聲作用對鼠大腦皮層超微結構的影響[J];第四軍醫(yī)大學學報;2002年09期

4 魏亞寧,劉靜,舒青,黃曉峰,陳景藻;次聲暴露對小鼠卵巢細胞超微結構的影響[J];第四軍醫(yī)大學學報;2003年04期

5 莊志強,裴兆輝,陳景藻;次聲生物學效應的相關機制[J];疾病控制雜志;2005年04期

6 魏亞寧,劉靜,舒青,黃曉峰,陳景藻;次聲暴露對小鼠睪丸細胞超微結構的影響[J];中華男科學;2002年05期

7 張國鋒;尹麗鶴;杜芳;黃旌;劉文博;謝克亮;趙鋼;;16Hz 130dB次聲預處理對大鼠局灶性腦缺血的保護作用[J];中華神經(jīng)外科疾病研究雜志;2009年06期

8 陳凱;曹煒;王階;;關于提高骨髓間充質(zhì)干細胞移植存活率的思考[J];現(xiàn)代中西醫(yī)結合雜志;2010年31期

9 袁華,龍華,李玲,牟翔,邱建勇,劉靜,陳景藻;次聲作用后大鼠海馬星形膠質(zhì)細胞的變化[J];中國臨床康復;2002年23期

10 王斌;陳景藻;劉靜;郭國珍;;次聲波對成骨樣細胞生物學特性的影響[J];中國臨床康復;2006年25期

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