CN113717930A - Cranial carotid interlayer specific induced pluripotent stem cell line carrying FBN1 mutation - Google Patents
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Abstract
The invention relates to a cranial carotid interlayer specific induced pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation, belonging to the technical field of biological medicines. The cell line is a human induced multifunctional stem cell line which carries FBN1 gene c.1858C > T mutation and is specific to the carotid mesentery; has been preserved in China Center for Type Culture Collection (CCTCC) at 09.01.2021 with the preservation number of CCTCC NO: C2021229. The establishment of the cell line provides a cell model for familial hereditary CAD caused by gene mutation; provides a cell model for the new drug screening and the drug treatment mechanism research of clinical cranial and carotid artery interlayer diseases; the cell resources of hereditary diseases are saved, and the disease-specific hiPSC cell bank is enriched and reserved for subsequent researches.
Description
Technical Field
The invention relates to a cranial carotid artery interlayer specific induced pluripotent stem cell line carrying FBN1 mutation, belonging to the technical field of biological medicines.
Background
The cranial carotid artery interlayer CAD (CAD) refers to laceration of intima and media or media and adventitia of intracranial and cervical artery vessels caused by various reasons, which causes blood to enter between artery walls through lacerated parts to form intercalary hematoma, and if thrombosis is formed, cerebral artery embolism and even occlusion are caused. The cranial-carotid artery interlayer is one of the most common causes of ischemic stroke in young people. Epidemiological studies have found that 10-25% of patients with first ischemic stroke under the age of 45 are caused by the cranial-carotid artery interlayer; furthermore, the true incidence may be higher since part of the arterial dissection does not cause any clinical symptoms or only causes mild non-specific symptoms. The pathogenesis of the cranial-carotid artery interlayer is quite complex, the disease is considered to be the result of the combined action of environmental factors and genetic factors, and the monogenic hereditary connective tissue diseases which are clearly related to the cranial-carotid artery interlayer at present comprise Ehlers-Danlos syndrome (type IV), myofibrodysplasia, Marfan syndrome and the like.
At present, medical research aiming at a cranial-carotid artery interlayer is mainly carried out on the level of an animal model, and due to species difference, the animal model and human have great difference on a pathophysiological system, and a plurality of disease mechanism researches and drug screening results cannot be directly applied to the human. Therefore, establishing a human-derived in vitro cell model is very important for researching the pathogenic mechanism of the cranial and carotid artery interlayer and exploring a treatment means.
Induced Pluripotent Stem Cells (iPSCs) four transcription factors (c-Myc, Oct3/4, SOX2, and Klf4) were introduced into mouse fibroblasts by Takahashi and Yamanaka (Takahashi et al,2006) by retrovirus and reprogrammed to form Embryonic Stem cell (ES cell) -like cells in 2006; human induced Pluripotent Stem Cells (hiPSCs) were established in 2007 by reprogramming human fibroblasts by introducing the same four transcription factors (c-Myc, Oct3/4, SOX2, and Klf4) and another set of transcription factors (Oct3/4, SOX2, Nanog, Lin28) to form ES-like Cells (Takahashi et al,2007, Yu et al, 2007). The hiPSCs technology provides a powerful driving force and revolutionary mode transition for transformation medicine and regenerative medicine, and also provides a cell source for the mechanism research of human genetic diseases.
In the research of hereditary cardiovascular diseases such as congenital cardiovascular diseases and hypertrophic cardiomyopathy, human cardiovascular pathological tissue specimens can hardly be obtained for in vitro research, and tissue cells such as blood vessels and cardiac muscles can not be cultured in vitro for a long time, so that pathogenic mechanisms of the diseases can be clarified, or a model for screening medicaments can be prepared. The construction of patient-specific ipscs can bypass the limitations of transgenic animal models in disease studies, such as dilated cardiomyopathy and long QT syndrome (Carvajal-Vergara et al, 2010; Itzhaki et al, 2011; Moretti et al, 2010; Narsinh et al, 2011 a; Sun et al, 2012; Yazawa et al, 2011), which successfully establish disease-specific ipscs and are used to simulate a variety of genetic cardiovascular diseases in vitro.
Through early clinical research, more than 100 sporadic samples are obtained for a cranial carotid artery interlayer patient, exon capture sequencing is utilized, and the fact that two cranial carotid artery interlayer patient FBN1 genes without genetic relationship and with highly similar clinical phenotypes carry c.1858C > T mutation is discovered, and at present, relevant literature reports that the site has CAD pathogenicity are temporarily absent, and the site belongs to a new pathogenic mutation site. One of the patients was reprogrammed to patient-specific iPSC using the episomal method, and no patent application for a cranio-carotid artery interlayer specific induced human pluripotent stem cell line carrying FBN1 gene c.1858c > T mutation was found internationally.
Disclosure of Invention
The invention aims to provide a cranial carotid interlayer specificity induced human pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation and a construction method thereof, the constructed cranial carotid interlayer specificity induced human pluripotent stem cell line containing FBN1 gene c.1858C > T mutation can solve the technical problem of providing biological materials for pathogenesis research and treatment research of FBN1 mutation-related diseases.
In order to solve the problems, the technical scheme adopted by the invention is to provide a cranial carotid interlayer specific induced human pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation, wherein the cell line is a cranial carotid interlayer specific induced human pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation; has been preserved in China center for type culture Collection (CGMCC) at 09.01.2021, the name of the culture is human induced pluripotent stem cells hIPSC/GD09006, and the preservation number is CCTCC NO: C2021229.
The invention provides application of a cranial carotid interlayer specificity induced human pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation in constructing a cranial carotid interlayer disease model.
The invention provides application of a cranial carotid interlayer specificity induced human pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation in preparation of a medicament for treating cranial carotid interlayer diseases.
The invention provides an application of a cranial carotid interlayer specificity induced human pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation in preparation of a diagnostic kit for diagnosing cranial carotid interlayer diseases.
The invention provides application of a cranial carotid interlayer specificity induced human pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation in screening or evaluating drugs for treating the cranial carotid interlayer.
The invention provides application of a cranial carotid interlayer specific induced pluripotent stem cell line (a cranial carotid interlayer specific induced human pluripotent stem cell line) carrying FBN1 gene c.1858C > T mutation in a detection kit for screening or evaluating the cranial carotid interlayer.
Compared with the prior art, the invention has the following beneficial effects:
1. the cranial carotid artery interlayer disease (CAD) specific induced multifunctional stem cell line carrying FBN1 gene c.1858C > T mutation is constructed, and a cell model is provided for familial hereditary CAD caused by gene mutation;
2. the establishment of the cell line can provide a cell model for the new drug screening and the drug treatment mechanism research of clinical cranial-carotid sandwich diseases, and provides an effective tool for the pathogenic mechanism research of the cranial-carotid sandwich diseases;
3. the invention saves the cell resource of hereditary diseases, enriches the disease-specific hiPSC cell bank and reserves for the subsequent research.
Preservation information
The culture is preserved in China Center for Type Culture Collection (CCTCC) at 09/01/2021, and the addresses of the China center for type culture Collection are as follows: eight-way Wuhan university 299 in Wuchang area, Wuhan university Collection in Wuhan City, Hubei province, zip code: 430072, culture name is human induced pluripotent stem cell hIPSC/GD09006, and preservation number is CCTCC NO: C2021229.
Drawings
Fig. 1 is a flow chart of hiPSC preparation.
FIG. 2 is a photograph of a patient DNA sequencing analysis electrophoresis; successfully constructing a sequencing peak picture of the hIPSC/GD09006 gene mutation c.1858C > T generation of the stem cells.
The figure shows that the stem cell hIPSC/GD09006 carries FBN1 gene c.1858C > T mutation.
FIG. 3 is a phase contrast light microscope of clone hIPSC/GD09006 stem cell line;
when the clone of the cell line hIPSC/GD09006 grows to a proper size, the clone continues to be passaged and amplified until the hipSC clone is stable, the cells grow like colonies and gather into a clone shape, the cells are small, the nucleus is large, the nucleus-to-mass ratio is high, the clone surface is smooth and flat, the edges are neat, and no dissociative cells exist.
FIG. 4 shows CytotUneTMiPS 2.0 Sendai reprogramming schematic diagram.
FIG. 5 is a graph showing the identification of stem cells hIPSC/GD09006 hipSC cell surface markers;
wherein the hiPSC surface Marker Oct4 fluorescence staining pattern is shown in fig. 5A, where > 90% positive results are passed; in FIG. 5A, Oct4 (nucleoprotein) labeled with red fluorescence, hoechst-labeled nucleus showed blue fluorescence under a fluorescence microscope, and cells showing blue-violet fluorescence after merge were positive cells.
The fluorescence staining pattern of hiPSC surface Marker SSEA4 is shown in fig. 5B, where SSEA 4: positive > 90%, result is passed; in FIG. 5B, SSEA4 (cell membrane protein) labeled with green fluorescence is blue under a hoechst-labeled nuclear fluorescence microscope, and cells with the nuclei encapsulated by green fluorescence after merge are positive cells.
FIG. 6 is a graph showing the dry test results of stem cells hIPSC/GD 09006.
The figure shows an alkaline phosphatase staining pattern, and the dry detection result is as follows: the alkaline phosphatase staining rate is more than 90 percent, and the result is qualified.
FIG. 7 is a chart showing the results of karyotype detection of stem cells hIPSC/GD 09006.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings:
as shown in fig. 1-7, the invention provides a cranial carotid interlayer specific induced human pluripotent stem cell line carrying FBN1 gene c.1858c > T mutation, wherein the cell line is a cranial carotid interlayer specific induced human pluripotent stem cell line carrying FBN1 gene c.1858c > T mutation; has been preserved in China center for type culture Collection (CGMCC) at 09.01.2021, the name of the culture is human induced pluripotent stem cells hIPSC/GD09006, and the preservation number is CCTCC NO: C2021229.
The invention provides application of a cranial carotid interlayer specificity induced human pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation in constructing a cranial carotid interlayer disease model.
The invention provides application of a cranial carotid interlayer specificity induced human pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation in preparation of a medicament for treating cranial carotid interlayer diseases.
The invention provides an application of a cranial carotid interlayer specificity induced human pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation in preparation of a diagnostic kit for diagnosing cranial carotid interlayer diseases.
The invention provides application of a cranial carotid interlayer specificity induced human pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation in screening or evaluating drugs for treating the cranial carotid interlayer.
The invention provides application of a cranial carotid interlayer specificity induced human pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation in a detection kit for screening or evaluating and detecting a cranial carotid interlayer.
The construction method of the skull carotid artery interlayer specificity induced human pluripotent stem cell line containing FBN1 gene c.1858C > T mutation comprises the following steps:
collecting the C.1858C carrying FBN1 gene>Peripheral Blood Mononuclear Cells (PBMC) of a T-mutant patient with a cranial-carotid artery interlayer are resuspended in a PBMC complete culture medium, inoculated to a 24-well plate, replaced by a fresh PBMC complete culture medium half a day, and cultured for 3 days; using a CytotubeTM2.0 Sendai virus kit transduces cells and incubate overnight; collecting cells, centrifuging to remove Sendai virus, suspending with PBMC complete culture medium and transferring to 24-well plate, continuously culturing for 2 days, replacing culture medium with cell factor-free PBMC culture medium, and inoculating transduced cells on a vitronectin coated cell plate; culturing with cytokine-free PBMC culture medium for 2 days, and half-replacing with Essential 8TMCulturing in culture medium for 1 day; then the fresh essentials 8 are replaced every dayTMMedia and hiPSC clones were observed; when the clone size was appropriate, hiPSC monoclonals were picked out and undifferentiated monoclonals were transferred to a new matrigel-coated cell plate. And when the hiPSC clone grows to a proper size, continuing passage and amplification until the hiPSC clone is stable, and the cells grow and gather into a clone shape in a colony shape, are small, have large cell nuclei and high nuclear-to-mass ratio, and have smooth and flat clone surfaces, regular edges and no free cells. As shown in fig. 3.
The constructed hipscs were subjected to first-generation sequencing, and the FBN1 gene was found to contain c.1858c > T mutation, i.e., the obtained hipscs were cranial carotid intima-specific induced pluripotent stem cells containing FBN1 gene c.1858c > T mutation. The culture name was named human induced pluripotent stem cells hIPSC/GD 09006.
Example (b):
1. the preparation method of the cranial carotid interlayer specific induced pluripotent stem cell containing FBN1 gene c.1858C > T mutation comprises the following steps:
(1) PBMCs were extracted from blood of one of the cranial-carotid artery sandwich patients in the Huashan Hospital affiliated to the university of Compound Dan as sample storage. PBMCs were thawed in PBMC complete medium and plated into 24-well plates for 3 days with half a day change of fresh PBMC complete medium.
(2) Using a CytotubeTM2.0 Sendai Virus kit resuscitated PBMCs were transduced, incubated overnight, cells harvested and centrifuged to remove Sendai Virus. Cells were resuspended in PBMC complete medium and transferred to 24-well plates for 2 consecutive days. The medium was replaced with cytokine-free PBMC medium on day 3 and transduced cells were plated onto vitronectin-coated cell plates. Changing the culture medium of the PBMC without the cell factors every other day on 4-6 days. 7 th day half-changed Essential 8TMAnd (4) a culture medium.
(3) On 8-28 days, fresh Essential 8 is changed every dayTMCulture medium, observing hIPSC/GD09006 clone, picking out hIPSC/GD09006 monoclonal when clone size is proper, and transferring undifferentiated monoclonal into new substrate gelatin matrigel coated cell plate. When the hIPSC/GD09006 clone grows to a proper size, continuing passage and amplification until the hIPSC/GD09006 clone is stable, and obtaining the clone containing c.1858C>T-mutated cranial-carotid interlayers specifically induce pluripotent stem cells.
Extracting hIPSC/GD09006 genome DNA and carrying out quality inspection, designing specific primers according to FBN1 gene sequence, carrying out first generation sequencing, and the detection result shows that the cell strain FBN1 gene has c.1858C > T mutation. The cells obtained are the cranial carotid interlayer specificity induced pluripotent stem cells containing FBN1 gene c.1858C > T mutation. As shown in fig. 2.
Attached table 1: electrophoresis chart of DNA sequencing analysis of patients and the construction of successful CAD-hipSC (hIPSC/GD09006) generation sequencing peak chart show that CAD-hipSC (hIPSC/GD09006) carries FBN1 gene c.1858C > T mutation.
2. hIPSC/GD09006 cell surface marker identification
ipscs have the ability to self-renew in vitro and therefore the cells obtained upon induction can be identified at the cellular level by fluorescent staining of the cell surface, such as: oct4, Nanong, Sox2, SSEA1, SSE4 and the like. In general, hipscs (human ipscs) do not express SSEA1, but express hES cell-specific surface antigen, and in this example, two iPSC Marker markers Oct4 and SSEA4 are selected as identification indexes; as shown in fig. 5;
the staining procedure of the surface Marker of hIPSC/GD09006 is as follows:
(1) iPSC cultured in 24-well plates was washed with DPBS and fixed with 4% paraformaldehyde
(2) Soaking in membrane-breaking buffer at room temperature (this step is limited to the nucleoprotein Oct4, and cannot be used for the membrane protein SSEA4)
(3) Sealing with sealing solution at room temperature, cleaning with DPBS twice, adding anti-Oct 4 and SSEA4 antibody to dilute, incubating
(4) The DPBS is washed twice, and a diluent of Anti Mouse AF 488 (green light labeled) and Anti Rabit AF 555 IgG-PE (red light labeled) antibodies is added
(5) And (3) cleaning the DPBS twice, adding a Hoechst nuclear staining solution diluent for nuclear counterstaining, cleaning the DPBS twice, adding 1% BSA (bovine serum albumin) and taking a picture.
The results are shown in FIG. 5, hIPSC/GD09006 stains normally, the nuclear protein expresses the dryness marker Oct4, and the cell membrane protein expresses the dryness marker SSEA 4.
3. And (3) detecting the dryness of the cells:
BCIP/NBT is a commonly used substrate for alkaline phosphatase. BCIP is hydrolyzed under the catalysis of alkaline phosphatase to generate a strong reactive substrate, and the product reacts with NBT to form insoluble NBT-formazan with dark blue to blue-violet color. The iPSC can express tissue non-specific phosphatase, so the BCIP/NBT alkaline phosphatase chromogenic kit purchased from Shanghai Bintian biotechnology limited company is used for identifying the induced multifunctional stem cells hIPSC/GD 09006. The method comprises the following steps:
(1) after the cell sample is fixed, it is washed 3-5 times for 3-5 minutes each with a washing solution.
(2) And (3) sequentially adding the solutions according to the proportion in the table, and uniformly mixing to prepare the BCIP/NBT dyeing working solution:
| alkaline phosphatase color buffer | 3ml | 10ml |
| BCIP solution (300X) | 10μl | 33μl |
| NBT solution (150X) | 20μl | 66μl |
| BCIP/NBT dyeing working solution | 3.03ml | 10.1ml |
(3) And after the last washing is finished, removing the washing solution, and adding a proper amount of BCIP/NBT dyeing working solution to ensure that the sample can be fully covered.
(4) Incubate at room temperature in the dark for 5-30 minutes or longer (up to 24 hours) until the color is developed to the desired shade.
(5) The BCIP/NBT staining working solution is removed, and the color reaction is stopped by washing with distilled water for 1-2 times.
(6) After the completion of the color reaction, the resultant was stained with neutral red staining solution (neutral red staining solution) and observed. As shown in fig. 6.
4. Karyotype detection
Karyotyping is a process of pairing, numbering and grouping chromosomes of induced pluripotent stem cells according to the morphological characteristics and specifications of chromosomes inherent in human cells, and performing morphological analysis. The method comprises the following steps:
(1) collecting a 6-well plate cultured for 24-48 hr and having a size of hIPSC/GD09006 and about 70%, adding colchicine (400ug/mL), mixing, and continuously culturing at 37 deg.C and 5% CO2After incubation for 3 hours in the environment, the culture medium was discarded. 1ml of accutase pre-warmed at 37 ℃ was added and digested for about 5 minutes. The addition of 1ml of medium was terminated, the cells were collected by gentle pipetting, the cell suspension was aspirated into a 50ml centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded.
(2) Adding 12ml of 0.075M KCl hypotonic solution preheated at 37 ℃ for more than 30 minutes, and lightly blowing and uniformly mixing. Rest at 37 ℃ for 30 minutes. Slowly adding 1.5ml of fresh fixing solution (methanol: glacial acetic acid ═ 3:1) along the tube wall, gently blowing and uniformly mixing, standing for 2 minutes, centrifuging at 1000 rpm for 10 minutes, and removing the supernatant. Then adding 10ml of fresh stationary liquid along the tube wall, gently blowing and uniformly mixing, standing for 2 minutes, centrifuging for 10 minutes at 1000 rpm, and removing the supernatant. Then, 10ml of fresh fixative was added along the tube wall, gently blown and mixed, stood for 30 minutes, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Adding 1ml of the fixing solution, and gently mixing to prepare a cell suspension. Fixing liquid: 24ml (methanol glacial acetic acid 18:6)
(3) Sucking a small amount of cell suspension, dropping 2-3 drops on a glass slide soaked by ice water, blowing away, and air-drying. And (3) placing the specimen into Giemsa dye liquor, dyeing for 8 minutes, washing with water to remove loose color, and air-drying. Observing the number and the dispersion of the chromosome sample split phases under a microscope.
(4) And (3) after the conventional flaking, putting the specimen into an oven with the temperature of 70 ℃ for dry baking for 2 hours, and naturally cooling. Adding 5ml of 2.5% pancreatin solution into a dyeing vat, adding 45ml of normal saline, adjusting the pancreatin solution to be purple red (pH 6.8-7.2) by using 1N HCl or 1N NaOH and phenol red, and pre-heating at 37 ℃. And (3) putting the slide specimen into a pancreatin solution for treatment for 25-45 seconds, and continuously and lightly shaking the slide to ensure that the pancreatin has uniform action. As the number of samples to be processed increases, pancreatin is gradually consumed, and the action time of the pancreatin is gradually prolonged. The chromosome slide specimen is taken out, placed in physiological saline at a pre-temperature of 37 ℃, and then the slide is rinsed with distilled water (or gently thrown to remove excessive pancreatin). And (3) putting the slide specimen into a Giemsa dye solution preheated to 37 ℃ for dyeing for 5-10 minutes. Washing with tap water, and air-drying. And (4) observing through a microscope. As shown in fig. 7.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Claims (6)
1. A cranial carotid interlayer specificity induction human pluripotent stem cell line carrying FBN1 gene c.1858C > T mutation is characterized in that: the cell line is a human induced multifunctional stem cell line carrying FBN1 gene c.1858C > T mutation and specific to a carotid mesentery; has been preserved in China center for type culture Collection (CGMCC) at 09.01.2021, the name of the culture is human induced pluripotent stem cells hIPSC/GD09006, and the preservation number is CCTCC NO: C2021229.
2. The use of the craniocostal specifically-induced human pluripotent stem cell line carrying the c.1858C > T mutation of FBN1 gene of claim 1 in constructing a model of a craniocostal disease.
3. The use of the human pluripotent stem cell line specifically induced by the carotid intima-media layer carrying the FBN1 gene c.1858c > T mutation according to claim 1 for the preparation of a medicament for the treatment of a carotid intima-media disease.
4. The use of the craniocostal specific-induced human pluripotent stem cell line carrying the c.1858C > T mutation of FBN1 gene of claim 1 in the preparation of a diagnostic kit for the diagnosis of a craniocostal disease.
5. The use of the cranio-carotid interlayer specific induced human pluripotent stem cell line carrying the FBN1 gene c.1858C > T mutation of claim 1 in screening or evaluating drugs for treatment of the cranio-carotid interlayer.
6. The use of the cranio-carotid interlayer specific induced human pluripotent stem cell line carrying the FBN1 gene c.1858C > T mutation of claim 1 in screening or evaluating a detection kit for detecting a cranio-carotid interlayer.
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