重组人Aβ_（1-42）的制备及促进细胞凋亡作用的实验研究
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摘要
科学研究表明,阿尔兹海默病(Alzheimer's disease, AD)的主要病理特征是细胞外老年斑的形成和细胞内神经纤维缠结的集聚,而老年斑的主要组成成分β-淀粉样蛋白42 (P-amyloid peptide, Aβ1-42)具神经元毒性,是AD的主要致病原因。神经干细胞(Neural Stem Cells, NSCs)是一类能自我复制,具有多向分化潜能细胞。神经干细胞的体外分离、体外培养、诱导分化到移植治疗,为神经系统损伤、神经退行性病变的治疗提供了有效的途径。鉴于此,本研究围绕Aβ1-42对细胞(人神经干细胞和人神经胶质瘤U251细胞)研究进行了如下工作。
利用基因重组技术构建了毕赤酵母真核表达载体pPICZaC-hAβ1-42>电转化毕赤酵母X-33,筛选出能够稳定高效分泌表达重组人Aβ1-42 (rhAβ1-42)的工程菌。重组蛋白经SDS-PAGE、Western Blotting、ELISA分析,证明表达的rhAβ1-42具有生物学活性。对rhAβ1-42进行5L摇瓶水平发酵,并对其发酵条件进行优化,rhAβ1-42表达量达到210mg·L-1。建立了一种分离纯化rhAβ1-42的方法。
我们对人神经干细胞进行体外分离、诱导分化、体外传代培养,同时对原代获得的神经干细胞进行鉴定,观察rhAβ1-42对神经干细胞生长活力的影响。利用光镜、MTT、免疫荧光、透射电镜、DNA-ladder、流式细胞术等方法和技术,分析了rhAβ1-42对神经干细胞的生长曲线、细胞形态、超微结构和凋亡作用的影响。结果表明rhAβ1-42能够诱导神经干细胞凋亡,对其生长具有明显的抑制作用,其抑制作用可能是通过促进神经干细胞凋亡实现的。
利用光镜、MTT、免疫荧光、Western Blotting、流式细胞术等方法和技术,分析了rhAβ1-42对人神经胶质瘤细胞U251生长曲线、细胞形态和凋亡的影响。结果表明rhAβ1-42能诱导神经胶质瘤细胞U251的凋亡,对其生长具有明显的抑制作用。
本研究创新之处：1)对rhAβ1-42进行毕赤酵母工程菌的摇瓶水平发酵,优化表达条件,建立一种适合于小规模纯化rhAβ1-42的方法,纯度为93.8%。2)全面、系统地研究了rhAβ1-42对人神经干细胞和U251细胞的作用,证实rhAβ1-42能够抑制神经细胞增殖、诱导神经干细胞的分化和凋亡。国内外未见相关报道。
Alzheimer's disease, first announced by the German researcher Alosi Alzheimer in 1907, gains more and more concern now. Amyloid-β(Aβ) is derived by the proteolytic processing of amyloid precursor protein(APP), resulting in a peptide pre-minantly 40 or 42 amino acids in length. Recently, studies on molecule biology and cytochemical involved senile plaques and neurofibrillary tangles have great progress. The latest results showed that the main pathologic characters of AD are extracellular senile plaques'formation and neurofibrillary tangles'accumulation in vivo. (3-amyloid peptide (Aβ1-42) which is a peptide of relative molecular mass about 4200 D. Aβ1-42 was composed of 39-43 amino acids, which hadβ-collapse peptides, and were calledβ-amyloid peptides.
Neural stem cells, which can produce nervous tissue or is derived of nervous system with self-renewal and make difference of in-house cells by asymmetry splitting. People think that the regenerate of neuron is not long to stop in the period of prenatal or postnatal ever since. When the neurons were damage or lesion, because of the mature neurons with disdivision growth, the nerve tissue could not be repaired completely and result in defecting with neurological function. However, the discovery of neural stem cells corrected this old traditionary viewpoint and the fact of its multi-directional differentiation also clear the debate on the source of neuron and glial cells problem since a long time. We have new recognition about the growth of neural system, the regenerate of neurons and the effect of neural stem cells on neural system disease or damage repair. The neural stem cells's biocharacteristics make it not only to become study on neural development and useful target on regenerate mechanism but also the neural stem cells embedding the neural system make it easy to survival and differentiation. It makes the cell transplantation to repair neural tissual lesion and to heal neural system disease's ideal cell category.
The neuroglioma is well-known on high-lethality rate. It usual grow in the deeply central neural system and the clinically adoptive on the exairesis and radiotherapy are have patential risk on patient. Apoptosis is on the condition of physiology or pathology and it follow the in-house procedure and finish the vital procedure. The dynamic equilibrium of cell proliferation and apoptosis are necessary basal biology process on organisma multicellularis maintain its structure stable, internal environment and growth development. The research indicted that tumor occure is probably due to disturbance of cell proliferation and apoptosis, which lead to tomer cells infinite proliferate. U251 is neuroglioma, which has similar to neurons. To researchβ-amyloid peptide on neuroglioma has important meaning.
The contents of this paper are focused on the following parts:1) Constructing expression vector pPICZαC-rhAβ1-42 transforming it into Pichia pastoris via electroporation and screening the engineering strains secreting the protein at high levels steadily.2) Studying the shake flask scale fermentation process of rhAβ1-42, optimizing the main parameters, and creating a new method for small-scale purification of rhAβ1-42.3) Purified rhAβ1-42 was in vitro induced to research human embryo neural stem cells.4) Purified rhAβ1-42 was in vitro induced to research U251 neuroglioma.
1 Constructing, screening and identificating of Pichia pastoris engineering strains expressing rhAβ1-42 steadily at a high level
1.1 Screening and identificating of the transformed Pichia pastoris strains
Linearize the expression vector pPICZαC-rhAβ1-42 after confirmed by sequencing and transform it into Pichia pastoris X-33 via electroporation. Single clones were picked from YPD plates containing Zeocin. Then the genomic DNA of the transformed yeasts were extracted to perform PCR using the expression primers. Yeast clones tested positively by PCR were proliferated and then rhAβ1-42 was induced with 0.5% methanol. The supernatant of fermentation was identified by SDS-PAGE and Western Blotting and the highest level strain was screened. SDS-PAGE showed that molecule weight of rhAβ1-42 was 4200 which was commensurate with documents report.Western blotting showed that the recombinant rhAβ1-42 has the same immunogen as natural.
2 Studies on shake flask fermentation and purification process of rhAβ1-42
2.1 Shake flask fermentation of rhAβ1-42 in Pichia pastoris
There are many advantages as a kind of expression host of Pichia pastoris, and we use it to fermentation rhAβ1-42 in shake flask. The study was focused mainly on pH value, induced time and etc. The results indicated the fermentation broth of pH6.0 when high yield was achieved, the fermentation broth of pH below 4.0. when hardly expressed. After methanol induction for about 72 h the expression level of rhAβ1-42 peaked. The concentration of rhAβ1-42 in the broth can reached 210 mg·L-1
2.2 A new method to purify rhAβ1-42
The fermentation supernatant was purified with ammonium sulphate sediment, dialysis desalination and AKTA explorer anion-exchange chromatography, then the eluate was high purity rhAβ1-42. The degree of purity could be 93.8%.
3 Studies on human embryo neural stem cells by different state of rhAβ1-42
3.1 Separate and culture of neural stem cells
Origin of separate and culture of neural stem cells by incubation BrdU two days, the cells showed BrdU positive reaction, indicating that the neural stem calls have the reproductive activity in vitro. The neural stem cells express specific antigen Nestin positive reaction, which prove that neurospheron's mainly component is neural stem cells.
3.2 Compare of the effect on neural stem cells growth curve by different species of rhAβ1-42
To test the effect on neural stem cells growth curve at the concentration of 10μmol/L by AO, AF, AN group, the result showed that the proliferation inhibition effect on neural stem cells by rhAβ1-42 and AF group is higher than AO and AN groups. 3.3 The effect on neural stem cells growth curve using different concentration
of rhAβ1-42
As the concentration of AF group are 0.5μmol/L,1μmol/L,5μmol/L, it has inhibition effect on the cell growth, but showed no change as the time passes. The result showed that the effect of grouth inhibition is obvious when the concentration of rhAβ1-42 is 10μmol/L and 20μmol/L.
3.4 rhAβ1-42-induced cell apoptosis
After treatment for 24h by 10μmol/L and 20μmol/L AO group and AF group, we examined cell apoptosis with Annexin-V-FITC by flow cytometry. The results demonstrate that AO group of 10μmol/L and 20μmol/L, the apoptosis rate is (62.12±6.31)%, (60.99±5.43)% respectively. AF group of 10μmol/L and 20μmol/L, the apoptosis rate is (67.54±7.11)%, (63.21±5.66)% respectively. AN group of 10μmol/L and 20μmol/L, the apoptosis rate is (47.21±4.77)%, (52.03±4.81)%。The result demonstrate that 10μmol/L and 20μmol/L AN group, AO group and AF group induced apoptosis obvious; some cells occure late apoptosis and necrosis.
3.5 DNA agarose gel electrophoresis
After treatment for 24h by 5μmol/L,10μmol/L and 20μmol/L, there are 3 lanes have obvious dispertion ladder, but control group and 0.5μmol/L,1μmol/L groups have no obvious ladders.
3.6 Morphology and ultra-microstructure observation
The observation under light microscopy and transmission electronic microscopy showed that the cell morphology turned to rotundity and oval, and Permeability reduced. The volume of cell turned smaller, cytoplasm turned much more abundant, and nucleus turned smaller and occurred vacuole.
3.7 Neurogenesis effcet on neural stem cells
After treatment on the concentration of 10μmol/L 24h, no change is observed on the total number cells compared to control cells. After treatment on the concentration of 10μmol/L 24h, marked BrdU showed no change compared to the control cells. The result showed neural stem cells morphology is intact, bright, well aligned and clear. BrdU of control group decrease obviously in number and showed no NeuN positive. The result hint Aβ1-42 not only inhibite the cell proliferation but also not unable the neural stem cells differentiate to neurons in the BrdU proliferative phase.
4 Studies on human neuroglioma U251 by Aβ1-42
4.1 The effect on cell proliferation of U251
To test the effect on U251 cells growth curve at the concentration of 0.5-20 umol/L by rhAβ1-42, the result showed that the growth inhibitation rate increase obviously and 20μmol/L reach the peak.
4.2 Observe the cell apoptosis morphology by microscope and AO/EB
The observation under light microscopy and microscopy showed that the U251 cell have cell-cell junction porosity, increased mesenchymal, cell shrinkage, part of the cell ablate, floating in the medium. Early apoptotic cells are nucleus showed greenyellow fluorescence with dense to crescentiform or granular, which are situated in side incline. With the concentration of rhAβ1-42 and time extend, the apoptosis cell number increased and late apoptotic cells showed nucleus are orange, dense and deviated nucleus. Cellular necrosis showed that cell volume had increased asymmetric red flourescence and outline is unclear.
4.3 rhAβ1-42-induced cell apoptosis
After treatment for 24h by period of 0.5μmol/L to 20μmol/L, we examined cell apoptosis by flow cytometry. The results demonstrate that rhAβ1-42 showed apoptosis and when 20μmol/L reached to 40.2%.
4.4 Test the expression apoptosis protein of bax and bcl-2
After treatment for 48h by a period of 0.5μmol/L to 20μmol/L rhAβ1-42, we examined apoptosis protein by western blotting. The result showed bax expression heighten, but bcl-2 showed trend of down regulation.
In this study we screened the engineering strains secreting the protein at high levels steadily. Studying the 5L shake flask scale fermentation process of rhAβ1-42, optimizing the main parameters, and creating a new method for small-scale purification of rhAβ1-42, the yield is about 210 mg/L. Purified rhAβ1-42 was in vitro induced to research cells (human neural stem cells and U251 neuroglioma.) It is confirmed that rhAβ1-42 could inhibit neural cells proliferation, induce stem cells differentiated and apoptosis. Our studies build experimental data for further studies on mechanism of AD and clinical therapy by rhAβ1-42.
利用基因重组技术构建了毕赤酵母真核表达载体pPICZaC-hAβ1-42>电转化毕赤酵母X-33,筛选出能够稳定高效分泌表达重组人Aβ1-42 (rhAβ1-42)的工程菌。重组蛋白经SDS-PAGE、Western Blotting、ELISA分析,证明表达的rhAβ1-42具有生物学活性。对rhAβ1-42进行5L摇瓶水平发酵,并对其发酵条件进行优化,rhAβ1-42表达量达到210mg·L-1。建立了一种分离纯化rhAβ1-42的方法。
我们对人神经干细胞进行体外分离、诱导分化、体外传代培养,同时对原代获得的神经干细胞进行鉴定,观察rhAβ1-42对神经干细胞生长活力的影响。利用光镜、MTT、免疫荧光、透射电镜、DNA-ladder、流式细胞术等方法和技术,分析了rhAβ1-42对神经干细胞的生长曲线、细胞形态、超微结构和凋亡作用的影响。结果表明rhAβ1-42能够诱导神经干细胞凋亡,对其生长具有明显的抑制作用,其抑制作用可能是通过促进神经干细胞凋亡实现的。
利用光镜、MTT、免疫荧光、Western Blotting、流式细胞术等方法和技术,分析了rhAβ1-42对人神经胶质瘤细胞U251生长曲线、细胞形态和凋亡的影响。结果表明rhAβ1-42能诱导神经胶质瘤细胞U251的凋亡,对其生长具有明显的抑制作用。
本研究创新之处：1)对rhAβ1-42进行毕赤酵母工程菌的摇瓶水平发酵,优化表达条件,建立一种适合于小规模纯化rhAβ1-42的方法,纯度为93.8%。2)全面、系统地研究了rhAβ1-42对人神经干细胞和U251细胞的作用,证实rhAβ1-42能够抑制神经细胞增殖、诱导神经干细胞的分化和凋亡。国内外未见相关报道。
Alzheimer's disease, first announced by the German researcher Alosi Alzheimer in 1907, gains more and more concern now. Amyloid-β(Aβ) is derived by the proteolytic processing of amyloid precursor protein(APP), resulting in a peptide pre-minantly 40 or 42 amino acids in length. Recently, studies on molecule biology and cytochemical involved senile plaques and neurofibrillary tangles have great progress. The latest results showed that the main pathologic characters of AD are extracellular senile plaques'formation and neurofibrillary tangles'accumulation in vivo. (3-amyloid peptide (Aβ1-42) which is a peptide of relative molecular mass about 4200 D. Aβ1-42 was composed of 39-43 amino acids, which hadβ-collapse peptides, and were calledβ-amyloid peptides.
Neural stem cells, which can produce nervous tissue or is derived of nervous system with self-renewal and make difference of in-house cells by asymmetry splitting. People think that the regenerate of neuron is not long to stop in the period of prenatal or postnatal ever since. When the neurons were damage or lesion, because of the mature neurons with disdivision growth, the nerve tissue could not be repaired completely and result in defecting with neurological function. However, the discovery of neural stem cells corrected this old traditionary viewpoint and the fact of its multi-directional differentiation also clear the debate on the source of neuron and glial cells problem since a long time. We have new recognition about the growth of neural system, the regenerate of neurons and the effect of neural stem cells on neural system disease or damage repair. The neural stem cells's biocharacteristics make it not only to become study on neural development and useful target on regenerate mechanism but also the neural stem cells embedding the neural system make it easy to survival and differentiation. It makes the cell transplantation to repair neural tissual lesion and to heal neural system disease's ideal cell category.
The neuroglioma is well-known on high-lethality rate. It usual grow in the deeply central neural system and the clinically adoptive on the exairesis and radiotherapy are have patential risk on patient. Apoptosis is on the condition of physiology or pathology and it follow the in-house procedure and finish the vital procedure. The dynamic equilibrium of cell proliferation and apoptosis are necessary basal biology process on organisma multicellularis maintain its structure stable, internal environment and growth development. The research indicted that tumor occure is probably due to disturbance of cell proliferation and apoptosis, which lead to tomer cells infinite proliferate. U251 is neuroglioma, which has similar to neurons. To researchβ-amyloid peptide on neuroglioma has important meaning.
The contents of this paper are focused on the following parts:1) Constructing expression vector pPICZαC-rhAβ1-42 transforming it into Pichia pastoris via electroporation and screening the engineering strains secreting the protein at high levels steadily.2) Studying the shake flask scale fermentation process of rhAβ1-42, optimizing the main parameters, and creating a new method for small-scale purification of rhAβ1-42.3) Purified rhAβ1-42 was in vitro induced to research human embryo neural stem cells.4) Purified rhAβ1-42 was in vitro induced to research U251 neuroglioma.
1 Constructing, screening and identificating of Pichia pastoris engineering strains expressing rhAβ1-42 steadily at a high level
1.1 Screening and identificating of the transformed Pichia pastoris strains
Linearize the expression vector pPICZαC-rhAβ1-42 after confirmed by sequencing and transform it into Pichia pastoris X-33 via electroporation. Single clones were picked from YPD plates containing Zeocin. Then the genomic DNA of the transformed yeasts were extracted to perform PCR using the expression primers. Yeast clones tested positively by PCR were proliferated and then rhAβ1-42 was induced with 0.5% methanol. The supernatant of fermentation was identified by SDS-PAGE and Western Blotting and the highest level strain was screened. SDS-PAGE showed that molecule weight of rhAβ1-42 was 4200 which was commensurate with documents report.Western blotting showed that the recombinant rhAβ1-42 has the same immunogen as natural.
2 Studies on shake flask fermentation and purification process of rhAβ1-42
2.1 Shake flask fermentation of rhAβ1-42 in Pichia pastoris
There are many advantages as a kind of expression host of Pichia pastoris, and we use it to fermentation rhAβ1-42 in shake flask. The study was focused mainly on pH value, induced time and etc. The results indicated the fermentation broth of pH6.0 when high yield was achieved, the fermentation broth of pH below 4.0. when hardly expressed. After methanol induction for about 72 h the expression level of rhAβ1-42 peaked. The concentration of rhAβ1-42 in the broth can reached 210 mg·L-1
2.2 A new method to purify rhAβ1-42
The fermentation supernatant was purified with ammonium sulphate sediment, dialysis desalination and AKTA explorer anion-exchange chromatography, then the eluate was high purity rhAβ1-42. The degree of purity could be 93.8%.
3 Studies on human embryo neural stem cells by different state of rhAβ1-42
3.1 Separate and culture of neural stem cells
Origin of separate and culture of neural stem cells by incubation BrdU two days, the cells showed BrdU positive reaction, indicating that the neural stem calls have the reproductive activity in vitro. The neural stem cells express specific antigen Nestin positive reaction, which prove that neurospheron's mainly component is neural stem cells.
3.2 Compare of the effect on neural stem cells growth curve by different species of rhAβ1-42
To test the effect on neural stem cells growth curve at the concentration of 10μmol/L by AO, AF, AN group, the result showed that the proliferation inhibition effect on neural stem cells by rhAβ1-42 and AF group is higher than AO and AN groups. 3.3 The effect on neural stem cells growth curve using different concentration
of rhAβ1-42
As the concentration of AF group are 0.5μmol/L,1μmol/L,5μmol/L, it has inhibition effect on the cell growth, but showed no change as the time passes. The result showed that the effect of grouth inhibition is obvious when the concentration of rhAβ1-42 is 10μmol/L and 20μmol/L.
3.4 rhAβ1-42-induced cell apoptosis
After treatment for 24h by 10μmol/L and 20μmol/L AO group and AF group, we examined cell apoptosis with Annexin-V-FITC by flow cytometry. The results demonstrate that AO group of 10μmol/L and 20μmol/L, the apoptosis rate is (62.12±6.31)%, (60.99±5.43)% respectively. AF group of 10μmol/L and 20μmol/L, the apoptosis rate is (67.54±7.11)%, (63.21±5.66)% respectively. AN group of 10μmol/L and 20μmol/L, the apoptosis rate is (47.21±4.77)%, (52.03±4.81)%。The result demonstrate that 10μmol/L and 20μmol/L AN group, AO group and AF group induced apoptosis obvious; some cells occure late apoptosis and necrosis.
3.5 DNA agarose gel electrophoresis
After treatment for 24h by 5μmol/L,10μmol/L and 20μmol/L, there are 3 lanes have obvious dispertion ladder, but control group and 0.5μmol/L,1μmol/L groups have no obvious ladders.
3.6 Morphology and ultra-microstructure observation
The observation under light microscopy and transmission electronic microscopy showed that the cell morphology turned to rotundity and oval, and Permeability reduced. The volume of cell turned smaller, cytoplasm turned much more abundant, and nucleus turned smaller and occurred vacuole.
3.7 Neurogenesis effcet on neural stem cells
After treatment on the concentration of 10μmol/L 24h, no change is observed on the total number cells compared to control cells. After treatment on the concentration of 10μmol/L 24h, marked BrdU showed no change compared to the control cells. The result showed neural stem cells morphology is intact, bright, well aligned and clear. BrdU of control group decrease obviously in number and showed no NeuN positive. The result hint Aβ1-42 not only inhibite the cell proliferation but also not unable the neural stem cells differentiate to neurons in the BrdU proliferative phase.
4 Studies on human neuroglioma U251 by Aβ1-42
4.1 The effect on cell proliferation of U251
To test the effect on U251 cells growth curve at the concentration of 0.5-20 umol/L by rhAβ1-42, the result showed that the growth inhibitation rate increase obviously and 20μmol/L reach the peak.
4.2 Observe the cell apoptosis morphology by microscope and AO/EB
The observation under light microscopy and microscopy showed that the U251 cell have cell-cell junction porosity, increased mesenchymal, cell shrinkage, part of the cell ablate, floating in the medium. Early apoptotic cells are nucleus showed greenyellow fluorescence with dense to crescentiform or granular, which are situated in side incline. With the concentration of rhAβ1-42 and time extend, the apoptosis cell number increased and late apoptotic cells showed nucleus are orange, dense and deviated nucleus. Cellular necrosis showed that cell volume had increased asymmetric red flourescence and outline is unclear.
4.3 rhAβ1-42-induced cell apoptosis
After treatment for 24h by period of 0.5μmol/L to 20μmol/L, we examined cell apoptosis by flow cytometry. The results demonstrate that rhAβ1-42 showed apoptosis and when 20μmol/L reached to 40.2%.
4.4 Test the expression apoptosis protein of bax and bcl-2
After treatment for 48h by a period of 0.5μmol/L to 20μmol/L rhAβ1-42, we examined apoptosis protein by western blotting. The result showed bax expression heighten, but bcl-2 showed trend of down regulation.
In this study we screened the engineering strains secreting the protein at high levels steadily. Studying the 5L shake flask scale fermentation process of rhAβ1-42, optimizing the main parameters, and creating a new method for small-scale purification of rhAβ1-42, the yield is about 210 mg/L. Purified rhAβ1-42 was in vitro induced to research cells (human neural stem cells and U251 neuroglioma.) It is confirmed that rhAβ1-42 could inhibit neural cells proliferation, induce stem cells differentiated and apoptosis. Our studies build experimental data for further studies on mechanism of AD and clinical therapy by rhAβ1-42.
引文
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