低声压次声对大鼠骨髓间充质干细胞活性的影响
发布时间：2018-08-29 15:19
【摘要】：背景 脑血管疾病(Cerebral vascular disease, CVD)是指由各种病因使脑血管发生意外而导致脑功能缺损的一组疾病的总称。脑血管疾病以其高发病率、高病死率和高致残率严重影响着人们的生存及生活质量,是多数国家的三大致死疾病之一。如何预防和治疗脑血管疾病与及怎样最大限度的降低脑血管疾病引起身体残疾成为研究热点,特别是当脑血管病发生后如何降低残疾率就变的至关重要了。目前临床上用于治疗脑血管病后遗症的方法众多包括神经营养因子、物理因子治疗和运动功能锻炼等方法,但仍然没有一种方法可以很好地治疗脑血管病引起的残疾。随着干细胞工程的推进,越来越多的学者注意到干细胞在疾病的治疗当中具有重大的潜能,特别是运用干细胞修复受损组织,干细胞替代治疗在组织损伤、遗传缺陷、退行性等疾病中都有相关的研究,其中骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)是目前备受关注的一类组织干细胞。 间充质干细胞是具有多方向分化潜能的成体干细胞,来源于中胚层间充质,主要存在于全身结缔组织和器官间充质中,以骨髓间充质中含量最为丰富。在骨髓当中主要的干细胞为造血干细胞及其祖细胞,只有一部分为间充质干细胞,BMSCs一方面是造血诱导微环境的重要成员,具有促进造血细胞增殖和分化的作用,参与支持和调控造血,帮助造血细胞粘附和归巢；另一方面,在适当的环境下,BMSCs可以分化为中胚层的细胞分化,甚至跨越胚层向外胚层及内胚层来源的组织细胞分化。BMSCs容易获得具有高增殖性,故可以体外大量增殖保证了充足的干细胞来源；BMSCs可以自体增殖然后移植,可避免免疫排斥问题同时在伦理道德上也没有限制。正是由于BMSCs具有的这些特点,才使得BMSCs成为了干细胞的研究热点。 对于临床上的许多疾病都有相关的模型试验应用BMSCs移植进行治疗,包括心血管疾病、血液疾病、脊髓损伤、颅脑损伤和脑血管疾病等疾病。在脑血管疾病中脑卒中会给患者带来严重的后遗症,有许多研究都致力于如何运用BMSCs的移植治疗脑血管病的后遗症,相应的研究结果基本证实了BMSCs移植可以改善脑卒中的预后提高神经功能。尽管许多学者都认可BMSCs移植治疗脑血管病的存在有益作用,但在实际的运用中存在许多问题。BMSCs移植治疗脑缺血疾病存在的问题包括移植途径、移植细胞数、移植的时间窗、移植细胞的追踪等,另一个存在的更重要的问题就是骨髓间充质干细胞移植后存活率低的问题。组织缺血后,缺血区内脑缺血缺氧造成脑组织周围环境恶劣、微环境的改变；当BMSCs迁移入缺血区内后由于生存环境的改变会导致BMSCs死亡造成细胞的存活率低,如果移植途径不是定向移植再加上在循环过程中细胞的丢失,这样能进入脑损伤部位的有活性的干细胞就会很少,这也是限制了BMSCs在临床上推广应用的一个难题。 物理因子治疗脑血管病已经成为了重要的临床手段,并且得到了学者认可。然而目前临床上应用的物理因子都有其优势和不足,急需科研工作者对潜在的物理因子进行研究,使更多的物理因子优势互补从而尽可能的降低脑血管病患者的致残率。次声是频率在0.0001-20Hz之间的机械振动波,其广泛存在于自然界。强度高于90dB的次声已被研究证实对生物体具有损伤作用,强度低于90dB的次声则在安全域内,我们把强度低于90dB的次声称为低声压次声。对于低声压次声具有的相关生物学效应却鲜有研究,我们通过对低声压次声的前期研究发现低声压次声具有与高声压次声相反的生理学作用,低声压次声似乎对受损机体会存在治疗作用。已有生理学研究结果表明,人体的各种器官都有一个固定的振动频率,如头部8-12Hz、胸部为4-6Hz、腹部为6-9Hz、盆腔为6Hz、心脏为5Hz。这些固有振动频率都在次声频率范围内,而次声对机体作用的生物学机制就是能引起器官、组织的生物共振。对次声的生物学效应的研究也从器官、组织深入到细胞,目前对细胞的研究主要是探讨次声对细胞的增殖、细胞周期、凋亡等生物学行为的影响。本实验小组在前期的初步研究中,我们发现低声压次声体外作用骨髓间充质干细胞(BMSCs)60min后可明显的促进细胞增殖抑制细胞凋亡,此结果预示着低声压次声可以提高BMSCs的活性,为解决BMSCs移植后存活率低的问题提供了一种研究方法。 虽然我们的初步研究证明了低声压次声能降低BMSCs的凋亡率提高其增殖活性,然而我们的前期研究在设计上不够完善,为了更详细地了解低声压次声对BMSCs的影响及得到相关的干预参数,本实验的第一步完善低声压次声对BMSCs的生物学效应影响的研究,接着深入探讨低声压次声对BMSCs生物学效应的影响与存活素(survivin)之间的关系,从而了解低声压次声改变BMSCs生物学效应的相关分子学机制。 目的 深入研究低声压次声对BMSCs的凋亡与增值的影响,完善干预参数真实性和可靠性,同时探讨此影响与survivin之间的关系,进一步地了解低声压次声对BMSCs作用,从而为低声压次声与BMSCs的运用提供一些理论基础。 研究方法 1.细胞获取和培养：用颈椎脱位法处死SD大鼠,取大鼠后肢的股骨和胫骨去除两端的骨骺,打开骨髓腔。用注射器吸取5mlDMEM/F12培养基反复冲洗骨髓腔,将冲洗后的骨髓悬液离心,倒掉上清液,用含10%胎牛血清的DMEM/F12培养基10ml重悬细胞,采用全骨髓贴壁培养法获取细胞。 2. BMSCs的传代和纯化：利用差异贴壁培养法纯化细胞,细胞首次按1：3传代,第二次与第三次传代按1：2传代方法。每次传代后做好标记P1、P2、P3及日期等的标记,放入37℃、5%C02孵箱中培养。本实验使用的细胞为第三代处于对数生长期的细胞。 3.试验处理及指标检测： (1)台盼蓝染色：为了保证用于实验的细胞有较高的活性降低实验误差,在细胞进行次声处理前对各组使用台盼蓝染色方法进行细胞活力检测。 (2)增殖与凋亡检测：取P3代细胞,分试验组和对照组,其中试验组用次声分别干预in、90min、120min,对照组暴露空气中相同时间。处理结束后检测细胞的增殖与凋亡,重复三次。 (3) survivin定性及定量检测：取P3代细胞,分试验组和对照组,试验组用次声干预60min,结束后放入细胞孵箱中培养72h,然后免疫荧光及qRT-PCR检测细胞survivin的表达含量。 统计方法 采用spss13.0统计软件对数据进行分析,各组数据以(x±s)表示。OD值及凋亡率的检测采用方差分析,survivin表达量使用独立样本t检验分析,以P0.05作为统计学差异检验标准。 结果 1.两组培养的P3代细胞存活率都在95%以上,说明细胞活力尚好,可以进行下一步实验研究。细胞活性率=活细胞总数／(活细胞总数+死细胞总数)×100% 2.实验组的OD值：60min (1.560±0.075),90min (1.160±0.096),120min (0.930±0.069);对照组OD值：60min (1.239±0.025),90min (1.090±0.110),120min (0.926±0.027)。结果显示实验组60min的OD值显著大于对照组60min,有统计学差异P0.01,90min和120min在实验组与对照组中无统计学差异,实验组中60min、90min与120min三组存在统计学差异,60minOD值最大,结果有统计学差异。结果证明低声压次声干预BMSCs60min可使细胞存在最大的增殖活性。 3.实验组的凋亡率：60min(10.70±1.47),90min(21.33±1.93),120min (22.87±1.45)；对照组凋亡率值：60min(18.20±1.12),90min(22.93±1.79),120min (23.47±2.50)。实验组的凋亡率在60min显著低于对照组60min的凋亡率(P0.01),90min和120min在实验组与对照组中凋亡率都较高,在实验组60min、90min与120min三组中,60min组凋亡率最低,结果有统计学差异。结果证明次声干预BMSCs60min可显著降低细胞的凋亡率。 4.通过观察低声压次声对BMSCs增殖与凋亡的影响,我们发现低声压次声在60min不仅可以提高BMSCs增殖活性还可以降低细胞的凋亡率,基于此结果我们认为60min为低声压次声干预BMSCs的一个理想的时间参数,故检测survivin的研究只观察低声压次声干预BMSCs60min。免疫组化结果显示实验组的BMSCs表达的survivin荧光强度显著强于对照组,实验组survivin的阳性率细胞计数(59.9±6.1),对照组survivin的阳性率细胞计数(24.3±5.8),两者的差异有统计学意义(P0.05)。为了更准确地知道次声对BMSCs表达survivin的影响,我们采用qRT-PCR定量检测survivin的含量；实验组survivin的mRNA的相对含量(1.318±0.051),对照组survivin的mRNA的相对含量(0.966±0.034),有统计学差异P0.01。结果证明低声压次声可显著地提高BMSCs表达survivin。 结论 1.低声压次声可以促进BMSCs的增殖,降低细胞的凋亡,通过综合分析各个干预时间点的结果,我们发现低声压次声在60min对BMSCs的生物学作用最大,据此我们可以推断60min为最理想的时间干预参数。 2.低声压次声可以在促进BMSCs增殖抑制细胞凋亡的同时存活素的表达水平也明显提高,据此我们初步推断低声压次声促进BMSCs增殖、抑制其凋亡的可能机制为次声提高了BMSCs分泌survivin的能力,然而是否存在直接的相关关系还需进一步的研究。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743
本文编号：2211603
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743
【参考文献】
相关期刊论文 前10条
1 袁华,陈景藻,李玲,饶志仁,邱建勇;次声作用后大鼠脑中热休克蛋白70的表达和分布[J];第四军医大学学报;1998年06期
2 费舟,章翔,王晓峰,刘先珍,陈景藻,贾克勇;次声作用后大鼠血脑屏障的改变及意义[J];第四军医大学学报;1999年08期
3 叶琳,龚书明,黄晓峰,王枫,刘静,陈景藻;次声作用对鼠大脑皮层超微结构的影响[J];第四军医大学学报;2002年09期
4 魏亚宁,刘静,舒青,黄晓峰,陈景藻;次声暴露对小鼠卵巢细胞超微结构的影响[J];第四军医大学学报;2003年04期
5 庄志强,裴兆辉,陈景藻;次声生物学效应的相关机制[J];疾病控制杂志;2005年04期
6 魏亚宁,刘静,舒青,黄晓峰,陈景藻;次声暴露对小鼠睾丸细胞超微结构的影响[J];中华男科学;2002年05期
7 张国锋;尹丽鹤;杜芳;黄旌;刘文博;谢克亮;赵钢;;16Hz 130dB次声预处理对大鼠局灶性脑缺血的保护作用[J];中华神经外科疾病研究杂志;2009年06期
8 陈凯;曹炜;王阶;;关于提高骨髓间充质干细胞移植存活率的思考[J];现代中西医结合杂志;2010年31期
9 袁华,龙华,李玲,牟翔,邱建勇,刘静,陈景藻;次声作用后大鼠海马星形胶质细胞的变化[J];中国临床康复;2002年23期
10 王斌;陈景藻;刘静;郭国珍;;次声波对成骨样细胞生物学特性的影响[J];中国临床康复;2006年25期
，本文编号：2211603
本文链接：https://www.wllwen.com/yixuelunwen/shenjingyixue/2211603.html
最近更新
教材专著
