CN116218787A

CN116218787A - Immortalized human alveolus type II epithelial cell line and construction method and application thereof

Info

Publication number: CN116218787A
Application number: CN202310439572.9A
Authority: CN
Inventors: 张文健; 胡慧青; 陈丽; 娄晋宁
Original assignee: Suzhou Guanguo Biomedical Technology Co ltd
Current assignee: Suzhou Guanguo Biomedical Technology Co ltd
Priority date: 2023-04-23
Filing date: 2023-04-23
Publication date: 2023-06-06
Anticipated expiration: 2043-04-23
Also published as: CN116218787B

Abstract

The invention belongs to the field of biomedicine, and particularly discloses an immortalized human alveolus II type epithelial cell line and a construction method thereof, wherein the preservation number of the cell line is CCTCC NO: C2022245, the cell line carries SV40T antigen, TTF1 gene and C-Myc gene, and the cell line stably expresses the gene and protein of specific surface active protein C of human alveolus II type epithelial cells; after the cell line is passaged for more than 80 times, the phenotype and secretion function of the cells are maintained unchanged, and the immortalized human alveolus II type epithelial cell line can be used for in vitro production and preparation of humanized lung surfactant and related medicaments, and has wide clinical application value in treating respiratory distress syndrome caused by various causes.

Description

Immortalized human alveolus type II epithelial cell line and construction method and application thereof

Technical Field

The invention belongs to the field of biomedicine, and relates to an immortalized human alveolus II type epithelial cell line and a construction method thereof, and a humanized lung surface active substance prepared by the cell line in vitro and therapeutic application of the active substance in treating various respiratory distress syndromes.

Background

Alveolar type II epithelial cells are important cells distributed in alveoli, and have the main functions of secreting lung surfactant (Pulmonary Surfactant, PS) and important physiological functions in maintaining alveolar functions, regulating blood-gas exchange of the lung and the like. The lung surfactant is distributed on the surface of alveolar epithelial cells, and is a complex composed of lipids secreted by alveolar type II epithelial cells and surfactant proteins, wherein about 90% of the lung surfactant is phospholipids, and about 10% of the lung surfactant is surfactant proteins. The surfactant proteins comprise four types A, B, C, D, wherein SP-C is unique to alveolar type II epithelial cells and is an important marker for identifying alveolar type II epithelial cells. The lung surfactant has important physiological functions of reducing the surface tension of alveoli, maintaining the size of alveoli, improving the compliance of alveoli, preventing atelectasis and pulmonary edema, and the like, and plays a vital role in maintaining the functions of the lungs.

Studies have shown that congenital or secondary lung surfactant deficiency or abnormality is one of the most important pathogenesis leading to respiratory distress syndrome, and that supplementation with exogenous PS is widely used clinically in the treatment of neonatal, pediatric and adult respiratory distress syndrome caused by various etiologies, and has good therapeutic effects, being one of the most effective drugs for the treatment of such diseases.

At present, only two non-humanized PS preparations are approved to be marketed in China, and two medicines are extracted from animal lung tissues: a preparation, GUERSU (Curosurf) (pig lung phospholipid injection), is prepared from whole lung tissue of pig; another trade name is Kerisu (bovine lung surfactant for injection) extracted from the lung lavage fluid of newborn calves. Although these PS products extracted from animals have clinically significant efficacy, these drugs all suffer from several common drawbacks (1) the presence of heterologous proteins in the product increases the incidence of allergic reactions in patients during treatment; (2) Long-term therapeutic use in patients can produce antibodies against PS, resulting in reduced efficacy or loss of activity of these drugs; (3) Products derived from xenogeneic animal tissues all have biosafety problems. The adoption of the conventional biological quarantine not only increases the production cost and the difficulty of product approval, but also greatly limits the market application of the product.

Researchers at home and abroad have attempted to replace these naturally extracted PS products with semisynthetic PS, synthetic PS, and recombinant PS. The results show that none of these semisynthetic PS, synthetic PS and recombinant PS can achieve the surface tension lowering effect of the natural PS product, so that these alternative products have not been approved for use in the market until now. Therefore, the preparation of the humanized natural PS product has unique clinical curative effect and market application prospect.

Alveolar type II epithelial cells are the only tissue cells that synthesize and secrete pulmonary surfactants. Because of the difficulty in the source of human alveolar type II epithelial cells and the inability to proliferate indefinitely in vitro culture, humanized lung surfactant has not been developed to date with success and without a commercially available product, and thus the preparation of humanized PS has been a dream of scientists and a desire of clinicians. In recent years, with the research and development of cell immortalization technology, it has become possible to construct cells that do not proliferate or have weak proliferation in vitro into immortalized cell lines that can proliferate indefinitely in vitro using gene transfection technology.

Disclosure of Invention

The invention mainly aims to establish an immortalized human alveolus II type epithelial cell line by adopting a gene transfection technology, and is used as engineering cells for producing humanized lung surface active substances in vitro, and the humanized lung surface active substances are extracted and prepared from culture solution by in vitro large-scale cell culture.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an immortalized human alveolus II type epithelial cell line carrying SV40T antigen, TTF1 gene and c-Myc gene.

Furthermore, an immortalized human alveolar type II epithelial cell line, named IHPEC-II (Immortalized human type II Pulmonary Epithelial cell line), was deposited in China Center for Type Culture Collection (CCTCC) for

type

8 and 3 of 2022, and the preservation number is CCTCCNO: C2022245. The preservation address is the postal code 430072 of university of Chinese Wuhan.

The invention also provides a method for constructing an immortalized human alveolus type II epithelial cell line, which comprises the following steps:

(1) Isolated culture of primary human normal alveolar epithelial cells;

(2) Constructing recombinant vectors, namely respectively constructing plasmid vectors carrying SV40T antigen, TTF1 gene and c-Myc gene;

(3) Gene transfection of primary human alveolar cells three genes were transfected on primary cultured human alveoli;

(4) Sub-cell clone screening of human alveolus type II epithelial cells;

(5) Identification of immortalized human alveolar type II epithelial cell lines.

Further, the recombinant vector in step (2) is selected from a retroviral vector, a lentiviral vector or a CRISPR/CAS plasmid. Preferably, the recombinant vector is a lentiviral vector. The slow virus vector can effectively integrate exogenous genes into host chromosomes, thereby achieving the effect of permanently expressing target sequences. Can effectively infect various types of cells such as neuron cells, liver cells, tumor cells, endothelial cells, stem cells and the like in the aspect of infection capability, thereby achieving good gene therapy effect. For some cells which are difficult to transfect, such as primary cells, stem cells, non-differentiated cells and the like, the use of the lentiviral vector can greatly improve the transduction efficiency of the target gene, greatly increase the probability of integrating the target gene into the genome of the host cell, and conveniently and rapidly realize the long-term and stable expression of the target gene. It will be appreciated that the type of carrier is not limited thereto and may be adapted to specific needs. In addition, the vector may contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, and the like, as well as other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly-U sequences, etc.).

Furthermore, in order to realize large-scale production and product acquisition in industrial production, the invention also provides a method for large-scale culture of an immortalized human alveolus type II epithelial cell line in vitro, which comprises the following steps:

(1) Cell plane culture: cell lines according to 1-5 x 10 ⁴ /cm ² Inoculating in a cell factory at a density, culturing in a carbon dioxide incubator, and after the cells grow to 90% confluence, adopting trypsin solution for digestion to collect cell suspension;

(2) Three-dimensional culture of cell microcarrier, and mixing cell suspension with pre-swelled sterilized microcarrier suspension according to 2-5 x 10 ⁵ After mixing the individual cells/ml microcarrier ratio, inoculating the mixture into a bioreactor for culture, wherein the culture conditions are as follows: the temperature is 37 ℃, the dissolved oxygen is 30-65%, the pH value is 7.15+/-0.25, and the ventilation rate is 0.2fsl;

(3) Continuous collection of cell culture fluid: and after the immortalized human alveolar type II epithelial cells on the microcarriers grow to 35% saturation, starting to collect the cell culture fluid.

As a specific embodiment, the invention provides a product, which is prepared by preparing lung surfactant lyophilized powder from immortalized human alveolus type II epithelial cell culture solution, and specifically preparing the lung surfactant lyophilized powder by sequentially subjecting the immortalized human alveolus type II epithelial cell culture solution to low-temperature refrigeration, thawing, filtering and vacuum drying.

Further, the lyophilized powder can be used for preparing a humanized lung surfactant (PS), wherein the PS is directly used as a PS preparation after being extracted and purified from the lyophilized powder, or is combined with one or more pharmaceutically acceptable compositions to prepare the surface active material preparation, and optionally, the composition can be a pharmaceutical excipient such as lactose, sodium chloride and the like; may also be a lipid comprising a lipid selected from phospholipids or neutral lipids, optionally the phospholipids are selected from saturated and/or unsaturated phospholipids, optionally the phospholipids are selected from dipalmitoyl phosphatidylcholine DPPC, palmitoyl oleoyl phosphatidylcholine POPC, dipalmitoyl phosphatidylglycerol DPPG, palmitoyl oleoyl phosphatidylglycerol POPG and sodium salts thereof, etc.; optionally the neutral lipid is selected from palmitic acid, cholesterol, triglycerides and the like.

As some specific examples, the product of the cell line preparation of the present invention, the lyophilized powder, or the lung surfactant obtained after purification of the lyophilized powder, may be optionally used to form PS preparations in combination with other active proteins or active protein analogues, or may be prepared into medicaments in combination with other medicaments suitable for intratracheal administration, optionally the PS preparations or the medicament combinations may further comprise one or more selected from isotonic agents, pH adjusting agents, buffering agents, viscosity adjusting agents, and the like, in the form of a clinically acceptable preparation such as an aerosol inhalant, an airway instillation or an injection, etc., it is understood that the present invention essentially provides a raw material for the preparation of medicaments, which may be prepared in various forms, for the treatment and/or prevention of diseases related to deficiency or dysfunction of lung surfactant.

Further, the disease associated with a deficiency or dysfunction of pulmonary surfactant is selected from the group consisting of neonatal respiratory distress syndrome NRDS, secondary respiratory distress syndrome ARDS, and severe pneumonia, meconium aspiration syndrome, novel coronavirus pneumonia, chronic obstructive pulmonary disease, and the like induced PS deficiency or dysfunction.

In addition, due to the unique diffusion characteristic and the characteristic of dynamically regulating the surface tension of PS, the freeze-dried powder can also be used as a medicinal carrier for preparing a medicinal preparation for transtracheal administration, and the PS can be used as a carrier for promoting the efficient and uniform diffusion of the medicament in the lung.

In summary, the invention provides an immortalized human alveolus type II epithelial cell line, and a construction method and application thereof. The immortalized human alveolus II type epithelial cell line not only can be a brand new technical method for preparing the humanized lung surfactant by the cell culture technology in vitro, but also can make up for the blank of lack of the humanized lung surfactant at home and abroad, provides a unique cell model for researching the pathogenesis of clinical respiratory diseases, and provides an effective therapeutic drug for patients with respiratory distress syndrome caused by various causes.

The immortalized human alveolar type II epithelial cell line disclosed by the invention has the advantages that:

1. the immortalized human alveolus type II epithelial cell line established by the invention is not reported in the literature at home and abroad.

2. When the cell line is established, gene transfection is carried out on primary cells, so that the phenotype change of alveolar type II epithelial cells caused by in vitro subculture is avoided, namely, the gene modification is completed when the cells still maintain the primary phenotype.

3. The technical method for preparing the lung surfactant by culturing the immortalized human alveolus type II epithelial cell line in vitro on a large scale and extracting from the cell culture solution is a brand-new natural PS production technical method.

4. The humanized lung surfactant prepared by the invention is a humanized PS product which is lacking at home and abroad at present, and can provide effective therapeutic drugs for patients with respiratory distress syndrome caused by various causes.

5. The immortalized human alveolus type II epithelial cell line established by the invention provides a unique cell model for the physiological and disease pathogenesis principles of the respiratory system.

Drawings

FIG. 1 shows a cell morphology of human primary alveolar epithelial cells before and after transfection of an immortalized gene (A: primary cultured human alveolar epithelial cells, B: human alveolar epithelial cells after transfection of an immortalized gene).

FIG. 2 shows the cell morphology of human alveolar epithelial cells before and after subcellular cloning selection (A: selected subcellular clones, B: expanded subcellular clones).

FIG. 3 shows a comparative graph of proliferation capacity in vitro for different passages of a human alveolar type II epithelial cell line; (in particular, the growth curves of the 3 rd generation and the 80 th generation of the immortalized human alveolar type II epithelial cell line of the present invention are compared).

FIG. 4 is a graph showing the results of characterization of human alveolar type II epithelial cell lines by RT-PCR; (wherein:

a: RT-PCR detection of mRNA expression of the SV40T antigen gene of the immortalized human alveolar II type epithelial cell line; lanes 1-3 are beta-actin, lanes 4-6 are SV40T antigen, 3 lanes of each gene PCR product from left to right: a549 (human lung cancer cell line), 3 rd generation immortalized human alveolar type II epithelial cell line, 80 th generation immortalized human alveolar type II epithelial cell line;

b: RT-PCR detection of mRNA expression of immortalized human alveolus type II epithelial cell TTF1 gene; lanes 1-3 are beta-actin, lanes 4-6 are TTF1, 3 lanes of PCR products per gene from left to right: a549, 3 rd generation immortalized human alveolar type ii epithelial cell line, 80 th generation immortalized human alveolar type ii epithelial cell line;

c: RT-PCR detection of mRNA expression of c-Myc gene in immortalized human alveolar type II epithelial cells, beta-actin in lanes 1-3, c-Myc in lanes 4-6, 3 lanes of PCR products of each gene from left to right: a549, 3 rd generation immortalized human alveolar type ii epithelial cell line, 80 th generation immortalized human alveolar type ii epithelial cell line;

d: RT-PCR detection of mRNA expression of immortalized human alveolar II type epithelial cell surface active protein gene (SP-A, B, C, D); 1-3 is beta-actin, 4-6 is SP-A,7-9 is SP-B,10-12 is SP-C,13-15 is SP-D, 3 lanes of PCR products per gene are from left to right: a549, 3 rd generation immortalized human alveolar type ii epithelial cell line, 80 th generation immortalized human alveolar type ii epithelial cell line;

e: RT-PCR (reverse transcription-polymerase chain reaction) detection of mRNA (messenger ribonucleic acid) expression of functional genes related to PS synthesis of human alveolar type II epithelial cells; lanes 1-3 are beta-actin, lanes 4-6 are CCT, lanes 7-9 are HNF3, lanes 10-12 are SREBP1c, 3 lanes of PCR products per gene are from left to right: a549, 3 rd generation immortalized human alveolar type ii epithelial cell line, 80 th generation immortalized human alveolar type ii epithelial cell line.

FIG. 5 shows characterization of human alveolar type II epithelial cell lines-SP-C immunofluorescence staining.

Fig. 6: a graph of the detection result of the secretion of surface active substances by the human alveoli type II epithelial cell line and the reduction of surface tension by the crude extract thereof; (wherein: A: the case where phospholipid is secreted by the human alveolar type II epithelial cell line cultured in vitro; B: the case where surface tension is reduced by the crude PS obtained by using the human alveolar type II epithelial cell line of the present invention).

Detailed Description

The invention will be further described with reference to the accompanying drawings and the detailed description, which are given by way of illustration only and do not limit the scope of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The invention provides an immortalized human alveolus II type epithelial cell line, which is specifically named an immortalized human alveolus II type epithelial cell line IHPEC-II (Immortalized human type II Pulmonary Epithelial cell line), and the preservation number of the cell line IHPEC-II is CCTCC NO: C2022245. The preservation address is the postal code 430072 of university of Chinese Wuhan. The immortalized human alveolar type II epithelial cell line IHPEC-II was received by the China Center for Type Culture Collection (CCTCC) at 3 days of 8 months of 2022, and registered for storing for thirty years from the day, the storage is continued for five years after receiving a request for providing a culture sample before expiration, and the viability of the cell line IHPEC-II was detected by the China center for type culture collection at 9 days of 8 months of 2022, and the result is survival.

EXAMPLE 1 establishment of immortalized human alveolar type II epithelial cell lines

1. Isolation culture of primary human alveolar epithelial cells:

washing lung tissue with D-Hank's solution, placing in a glass plate, and cutting into 1mm pieces ³ Is digested for 10 minutes at 37℃using 10ml of 0.25% pancreatic protease solution, and then digested for 30 minutes at 37℃with 10ml of 0.1% type I collagenase, and then digested with 5ml of fetal bovine serum. After the cell digest was filtered through a 200 mesh cell sieve, the filtrate was centrifuged at 1600rpm for 8 minutes. Complete medium for precipitation (DMEM/F12 medium supplemented with 10% fetal bovine serum, 10% ^-8 M dexamethasone, 160U/L insulin) was resuspended and inoculated into 24-well cell culture plates at 37℃with 5% CO ₂ Culturing in incubator, and changing complete culture medium every 3 days.

2. Construction of lentiviral expression vectors:

a commercial third generation lentiviral vector was used, containing a total of 4 plasmids, derived from the vast plasmid platform, plasmid name and number: pLP1 (cat No. P0264), pLP2 (cat No. P0265), pLP-VSVG (cat No. P0266) and pRRLsin-SV40T anti-IRES-mCherry (cat No. P37188).

pRRLsin-SV40T anti-IRES-mCherry was used as an expression vector and modified using a seamless cloning kit (Shanghai Biyunshan Biotechnology Co., ltd., cat# D7010S) to obtain the final SV40T, TTF1 and c-Myc expression vectors. The method comprises the following steps:

2-1, construction of SV40T expression vectors: firstly, carrying out resistance transformation on the expression vector, using the seamless cloning kit, replacing the mCherry sequence of 6498-7208 in pRRLsin-SV40T anti-IRES-mCherry vector with puromycin resistance gene, and naming the purery sequence as pRRLsin-SV40T anti-IRES-Puro, wherein the coding sequence of SV40T antigen is referred to the CDS sequence of Genebank sequence number NC_001669.1, and the puromycin resistance gene sequence is referred to the puroR sequence of plasmid #131613 on addgene. Org.

2-2, construction of TTF1 expression vectors: using a seamless cloning technique, the mCherry sequence 6498-7208 in prrlin-SV 40T anti-IRES-mCherry was replaced with a blasticidin resistance gene-BSD gene sequence, designated prrlin-SV 40T anti-IRES-BSD, which is the BSD sequence of plasmid #124982 on addgene. Org. Then, the SV40T sequence 3716-5842 in pRRLsin-SV40T anti-IRES-BSD was replaced with TTF1 coding sequence, which was designated pRRLsin-TTF1-IRES-BSD, and this was the TTF1 expression vector used in the present invention, and the coding sequence of TTF1 was found in the CDS sequence of Genebank sequence No. NM-003317.4.

2-3, construction of c-Myc expression vector: the mCherry sequence of 6498-7208 in pRRLsin-SV40T anti-IRES-mCherry was replaced with the bleomycin resistance gene-Zeocin gene sequence, designated pRRLsin-SV40T anti-IRES-Zeocin, see BleoR sequence of plasmid #171685 on adedge. Org, using a seamless cloning technique. Then, the SV40T sequence 3716-5842 in pRRLsin-SV40T anti-IRES-Zeocin was replaced with the c-Myc coding sequence designated pRRLsin-c-Myc-IRES-Zeocin, which is the c-Myc expression vector used in the present invention, see plasmid #180278 c-Myc sequence on AddGene.

3. Packaging of lentiviral particles:

293T cells were transfected 24h before 10 ⁶ Is inoculated in a T25 cell culture flask, 3 flasks, and used for preparing lentiviral particles carrying SV40T, TTF1 and c-Myc, respectively. Fresh medium (DMEM/F12 medium with 10% fetal bovine serum) was changed 2h before transfection. 10.5ug of plasmid DNA was transfected per flask, including: 5 μg gene expression plasmid, 2 μg pLP1 plasmid, 2 μg pLP2 plasmid and 1.5 μg pLP-VSVG plasmid. The above DNA was mixed in 500ul of Opti-MEM solution to prepare a DNA mixture (solution A). Solution B was prepared by adding 31.5. Mu.g PEI to 500. Mu.l of Opti-MEM solution. Mixing A, B solutions, and standing at room temperature for 20min. The mixture was then added to the cultured 293T cells, gently mixed, labeled, and incubated in an incubator at 37℃for 0 hours. After 6 hours fresh medium was changed. Collecting culture supernatants of each bottle for 48 hr and 72 hr, mixing, centrifuging for 10min at 900g to remove cell debris, filtering with needle filter with 0.45 μm pore diameter to obtain three kinds of slow virus particles with SV40T antigen, TTF1 gene and c-Myc gene with infectious capacity but replication defect, and directly using or freezing the packaged virus particlesReserve at-80 ℃.

4. Viral transfection of cells:

firstly, 250. Mu.l each of virus suspension carrying SV40T antigen gene was added to a cell culture well containing 500. Mu.l of primary human alveolar epithelial cells in complete medium, and after 24 hours of infection, fresh complete medium was replaced (DMEM/F12 medium supplemented with 10% fetal bovine serum, 10% ^-8 M dexamethasone, 160U/L insulin), 3. Mu.g/ml puromycin was added after 3 days of incubation for 3 days during which cell death was seen, puromycin was removed after 3 days and fresh complete medium was changed for incubation. The proliferation of cells was accelerated after the SV40T antigen gene transfection, and the morphology changed from large to flat to small to three-dimensional before the gene transfection, as shown in FIG. 1. When the cells grow to about 50% confluence, mixing the virus suspension carrying TTF1 gene with fresh complete culture medium 1:1, changing liquid to 24-well plate, changing fresh complete culture medium after 24 hours (adding 10% fetal bovine serum and 10% fetal bovine serum into DMEM/F12 culture medium) ^-8 M dexamethasone, 160U/L insulin), 10 μg/ml blasticidin was added 3 days after culturing for continued culture for 3 days, during which time cell death was seen, blasticidin was removed 3 days later and fresh complete medium was replaced for expansion culture. When the cells grow to about 50% confluence, mixing the virus suspension carrying the c-Myc gene with fresh complete medium 1:1, changing the liquid to a 24-well plate, and changing the fresh complete medium after 24 hours (adding 10% fetal bovine serum and 10% fetal bovine serum into DMEM/F12 medium) ^-8 M dexamethasone, 160U/L insulin), after 3 days of culture, 10 μg/ml bleomycin was added for continued culture for 3 days, during which time cell death was seen, bleomycin was removed after 3 days, and fresh complete medium was changed for expansion culture.

5. Subcellular clone screening method for screening human alveolar type II epithelial cells:

cells transfected with the gene were transformed at 1X 10 ³ The low density inoculated T25 cell culture flask was used for culturing for 1 week, then sub-cell clone with typical alveolar type II epithelial cell morphology was selected under an inverted microscope, other cells around the cell clone were carefully scraped off under a microscope with a fine glass tube sterilized under an autoclave, and the growth of the cells was observed daily until the sub-clone cells grew over 50 cells [ ]In FIG. 2A), 1 drop of pancreatin is sucked by an elbow suction pipe to digest locally on the cell clone, after digestion, the cells are directly sucked into the elbow suction pipe, taken out and inoculated into a new 24-hole culture plate for continuous culture, and after growing and converging, the cells are continuously passaged and amplified to a 6-hole plate and then to a T25 cell culture flask. The obtained human alveolar type II epithelial cell line clone, cells were grown in polygonal shape and monolayer (B in FIG. 2).

Example 2 immortalization of the in vitro proliferation Capacity of the human alveolar type II epithelial cell line

To determine the in vitro proliferation capacity of the immortalized human alveolar type II epithelial cell line established in example 1, CCK-8 cell proliferation experiments were performed with the 3 rd and 80 th generation cells, respectively. Specifically, two-generation cells were inoculated into 96-well plates at the same cell density (2000 cells/well), 3 wells were taken at

days

0, 1, 4, 7, and 10 of culture, and CCK8 solution (bi yun tian, C0039) was diluted 10-fold with fresh medium and the wells to be measured were changed. After incubation in a incubator at 37℃for 4 hours, the absorbance of each well at 450nm was measured with an enzyme-labeled instrument. Cell growth was plotted using GraphPad 8.0 software according to absorbance values at different time points and the results were shown as mean ± SD. As a result, as shown in FIG. 3, the established immortalized human alveolar type II epithelial cell line proliferated rapidly with a doubling time of about 36 hours, and the in vitro proliferation capacities between the 3 rd and 80 th generation cells were not significantly different statistically (FIG. 3).

Example 3 phenotypic identification of immortalized human alveolar type II epithelial cell lines

The phenotype of the immortalized human alveolar type II epithelial cell line obtained in example 1 was evaluated, and A549 cells (human lung cancer cell line) were used as a control, and the immortalized human alveolar type II epithelial cell lines of the 3 rd and 80 th generations were used as subjects.

1) mRNA expression conditions of immortalized human alveolus II type epithelial cell line immortalized genes (SV 40T antigen), key function regulatory genes (TTF 1, C-Myc), alveolus II type epithelial cell surface active protein C (SP-C) and other functional genes are detected by using an RT-PCR method.

Specifically, the RNA extraction kit (Ai Kerui, AG 21022) was used to extract A549, 3 rd generationTotal RNA from the 8 th generation IHPEC-II cells was then reverse transcribed into cDNA using an RNA reverse transcription kit (Ai Kerui, AG 11728). PCR was performed using a PCR kit (Takara) to detect genes of 4 aspects: 1) Detecting transfected gene-SV 40T antigen capable of immortalizing cells to prove successful cell immortalization; 2) Detecting a key transcription factor TTF1 for controlling the phenotype of the alveolar type II epithelial cells and a gene c-Myc for inhibiting cell differentiation so as to determine that the gene regulation level has the characteristics of the alveolar type II epithelial cells; 3) Detecting specific surface active protein genes of the alveolar type II epithelial cells, including a marker protein SP-C thereof and other three surface active proteins SP-A, SP-B, SP-D, wherein the cell line is proved to be the alveolar type II epithelial cell line from the viewpoint of the marker protein; 4) Detecting CCT (CCT-related enzyme), HNF3 (surfactant protein-related transcription factor) and SREBP1c (lipid-related transcription factor) which are involved in phospholipid synthesis, so as to prove that the cell line has a phospholipid synthesis function; furthermore, the housekeeping gene β -actin was used as an internal reference. All primers were synthesized by Shanghai Biotechnology Inc., SV40T primers were designed using Primer Premier 5 software, SV40T product length was 199bp, other Primer sequences were referenced to PrimerBank website, and specific Primer ID numbers are shown in Table 1. The amplification conditions were: the initial denaturation temperature is 95 ℃ for 2min, the cyclic denaturation temperature is 95 ℃ for 30s, the annealing temperature is 58 ℃ for 30s, and the extension temperature is 72 ℃ for 30s, wherein the total period is 30 cycles. The final extension temperature was 72℃for 10min and cooled at 4 ℃. 6ul PCR products containing loading buffer (Biyundian, D0071) and Gelred (Soy pal, G5560) were subjected to 2% agarose gel electrophoresis in Bio-Rad Chemieoc ^TM Imaging analysis was performed on an xrs+ gel imaging system.

TABLE 1 PCR amplification primer information

Gene	PrimerBank primer ID number	Product length
			TTF1	4507715a1	68bp
c-Myc	239582723c1	119bp
			SP-A	13346504a1	101bp
SP-B	288856298c1	87bp
			SP-C	288915520c1	96bp
SP-D	61699225c3	186bp
			CCT	31543384c3	218bp
HNF3	340545541c3	175bp
			SREBP1c	256665250c1	222bp
β-actin	4501885a1	250bp

The results show that the 3 rd and 80 th generation immortalized human alveoli II type epithelial cell lines stably express the immortalized gene SV40T antigen (A in FIG. 4) and the key function regulatory genes TTF1 (B in FIG. 4) and C-Myc (C in FIG. 4); mRNA of the alveolar type II epithelial cell-specific surfactant gene SP-C is expressed, and SP-A, SP-B, SP-D is also expressed (D in FIG. 4), mRNA of a key enzyme involved in phospholipid synthesis CCT, a key transcriptional factor HNF3 for surfactant protein regulation and a key transcriptional factor SREBP1C for lipid synthesis regulation are expressed (E in FIG. 4).

2) The expression of SP-C specific to immortalized human alveolar type II epithelial cells was detected by immunofluorescence staining.

Specifically, IHPEC-II cells are inoculated on a cell climbing sheet, the climbing sheet is taken when the cell confluence reaches 80%, and the cell climbing sheet is fixed for 10 minutes by pre-cooled ice methanol at-20 ℃ after being washed by cold PBS. Washed 3 times with PBS and blocked with 10% goat serum for 1 hour at room temperature. SP-C primary antibody (Abclonal, A1835) was incubated overnight at 4℃and then the samples were washed and incubated with Alexa Fluor 594-labeled rabbit anti-goat IgG fluorescent secondary antibody (Thermo Fisher Co., cat# A-11005) for 1 hour. Nuclei were stained with DAPI (bezotai, C1002). Finally, after the tablet is encapsulated by the anti-fluorescence quenching encapsulation tablet, imaging is carried out under a fluorescence microscope. The results demonstrate that immortalized human alveolar type II epithelial cells also stably express SP-C at the protein level (FIG. 5).

The experimental result shows that the immortalized human alveolar type II epithelial cell line provided by the invention has the phenotypic characteristics of alveolar type II epithelial cells, and the phenotype of cells is still stable in the 3 rd generation to the 80 th generation.

EXAMPLE 4 immortalized human alveolar type II epithelial cell line production of Lung surfactant in vitro culture

Amplifying and culturing the 3 rd generation and 80 th generation immortalized human alveolus II type epithelial cell lines respectively, taking A549 cells as a control, changing the cells into serum-free phenol red-free DMEM/F12 culture medium when the cell confluence reaches 80%, and collecting culture supernatants of the A549 cells, the 3 rd generation and the 80 th generation immortalized human alveolus II type epithelial cells respectively after 24 hours.

1) The amount of phospholipids in the immortalized human alveolar type II epithelial cell line conditioned medium was measured using the Abcam phospholipid assay kit (Abcam, ab 234050).

The specific operation method is that after the reagent rewarms, the standard substance and the reaction mixture are prepared according to the operation instructions, and the absorbance at 570nm is measured after incubation for 30 minutes at room temperature. And drawing a standard curve, and calculating according to absorbance to obtain the content of the phospholipid.

The results show that the phospholipid content in the conditioned medium of the 3 rd generation and 80 th generation immortalized human alveolus II type epithelial cell line is respectively

147.9.+ -. 25.02mg/L and 149.9.+ -. 32.15mg/L, significantly higher than A549 cells (17.07.+ -. 0.71 mg/L), and there was no significant statistical difference in phospholipid content between the 3 rd and 80 th generation immortalized human alveolar type II epithelial cell lines (FIG. 6A).

2) Crude extracts of lung surfactant were extracted from immortalized human type ii epithelial cell cultures by centrifugation (3000 g x 15min for supernatant and then centrifugation 10000g x 60 min for pellet). The crude extract extracted from 1L of the culture broth was dissolved in 20ml of pure water as a sample, and the surface tension of the sample was analyzed by a surface tensiometer (Shanghai Fang Rui, BZY 200). The results showed that the crude extract of pulmonary surfactant reduced the surface tension by about 38.2% compared to the control (distilled water) (B in fig. 6).

EXAMPLE 5 establishment of in vitro Large Scale culture of immortalized human alveolar type II epithelial cell lines and production of humanized Lung surfactant

The invention adopts the following technical scheme:

(1) The technical method for establishing the large-scale planar culture immortalized human alveolus type II epithelial cell line of an in vitro cell factory comprises the following steps:

immortalized human alveolar type II epithelial cells after resuscitationIs according to 1-5 x 10 ⁴ /cm ² Preferably 2.5 x 10 ⁴ / cm ² Is suspended in a complete cell culture medium, and is inoculated into 4 10-layer cell factories, respectively, cells are cultured at 37 ℃ and 5% CO ₂ Culturing in an incubator, changing the culture medium every 3 days, and when the cells grow to 90% confluence, adopting trypsin solution for digestion, and collecting cell suspension for in-vitro large-scale microcarrier three-dimensional culture.

(2) The technical method for establishing the three-dimensional culture of the immortalized human alveolus II type epithelial cell microcarrier comprises the following steps:

mixing the cell suspension of immortalized human type II alveolar epithelial cells collected by a cell factory with the pre-swelled sterilized microcarrier suspension according to a certain proportion (2-5 x 10) ⁵ Individual cells/ml microcarrier), in a BioFlo 320 bioreactor from Eppendorf, the culture conditions were controlled at 37℃with dissolved oxygen of 30-65%, pH 7.15.+ -. 0.25 and aeration of 0.2fsl. Half of the culture solution is replaced every day, and when the immortalized human alveolar type II epithelial cells on the microcarriers grow to 35% saturation, the cell culture solution is collected.

(3) The technical method for continuously collecting the immortalized human alveolus II type epithelial cell culture solution in vitro is established:

when immortalized human alveolar type II epithelial cells on the microcarriers grow to 35% saturation, the cell culture fluid in the bioreactor starts to be collected, namely: half of the broth was collected daily and an equal amount of fresh broth was replenished and collected continuously for no more than 21 days.

EXAMPLE 6 technical method for preparing Lung surfactant lyophilized powder and purifying PS from immortalized human alveolar type II epithelial cell culture solution

The main technical method for preparing the freeze-dried powder comprises the following steps:

1) The collected immortalized human alveolus II type epithelial cell culture solution is collected in a 20 liter sterile medical plastic bucket and is placed in a refrigeration house at the temperature of minus 20 ℃ for preservation.

2) Thawing the culture solution with the required dosage, filtering the immortalized human alveolus II type epithelial cell culture solution by a filter membrane of 3000g for 15 minutes or 1.0 mu m to remove dead cells and cell fragments, centrifuging the supernatant at 10000g for 60 minutes at high speed to obtain precipitate which is the PS crude extract, re-suspending the precipitate with a small amount of distilled water solution, packaging into penicillin bottles, and placing into a vacuum freeze dryer to prepare freeze-dried powder through vacuum freeze-drying.

The main technical method for purifying the humanized lung surfactant comprises the following steps:

dissolving the above collected lyophilized powder with physiological saline, centrifuging with gradient of 0.65mol/L sucrose at 4deg.C for 30min at 20000g, collecting white precipitate of intermediate layer of physiological saline and sucrose, centrifuging with distilled water for 2 times, and collecting precipitate according to Folch method (FOLCH J, LEES M, SLOANE STANLEY GH. A simple method for the isolation and purification of total lipides from animaltissues).J Biol Chem1957;226 (1): 497-509.) PS was purified by dissolving the precipitate in physiological saline and adding 2 volumes of chloroform-methanol (2: 1, V/V), and after vortexing, was placed in a refrigerator at 4 ℃ overnight. After centrifugation at 700g for 10min at 4deg.C, the phase liquid was removed and dried with nitrogen. Adding 5 times of pre-cooled acetone, shaking, centrifuging and precipitating at-20deg.C for 4000 g/10 min to obtain milk white substance which is humanized PS pure product, and storing in a refrigerator at-20deg.C for use.

EXAMPLE 7 preparation of humanized Lung surfactant inhalant

3.0g of lactose as a pharmaceutical auxiliary material is weighed and dissolved in 200ml of pure water, and then 100mg of humanized PS pure product extracted according to the method described in example 6 is added and stirred for dissolution. The preparation micronization process adopts BuchiB-290 spray dryer to prepare inhalation powder spray according to operation instruction. Finally, collecting the dry powder, filling the dry powder into an inhaler to prepare the humanized PS inhalant, and storing the finished product in a refrigerator at the temperature of-20 ℃.

EXAMPLE 8 preparation method of humanized Lung surfactant tracheal drop

100mg of purified humanized PS purified by extraction according to example 6 is weighed, dissolved in 1.5ml of physiological saline, sterilized by a 0.22 μm filter membrane, aseptically filled in separate bottles to prepare an airway drop, and stored in a refrigerator at 4 ℃. The dripping agent can be injected into lung via fine plastic catheter after trachea cannula, and can be used for treating neonate RDS.

EXAMPLE 9 use of humanized PS as a pharmaceutically acceptable carrier in an intratracheal formulation

The humanized PS extracted in example 6 can be used as a pharmaceutical carrier for preparing an intratracheal administration preparation, and the method for preparing the medicament by combining PS with antibiotics or PS with hormone is as follows:

1) The gentamicin phospholipid emulsion is prepared by weighing 100mg of the humanized PS pure product described in the example 6, adding 4ml of physiological saline and 1ml of gentamicin sulfate injection (1 ml:40mg of Shijia Kagaku Co., ltd.) and mixing, and finally preparing into the emulsion containing gentamicin and PS with the concentration of 8mg/ml and 20mg/ml respectively.

2) 100mg of the humanized PS pure product described in example 6 is weighed and mixed with 1ml of dexamethasone sodium phosphate injection (1 ml:2 mg/branch of pharmaceutical Co., ltd.) to finally prepare a mixed solution containing dexamethasone and PS phospholipid with the concentration of 2mg/ml and 100mg/ml respectively.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

1. An immortalized human alveolar type ii epithelial cell line carrying the SV40T antigen, TTF1 gene and c-Myc gene.

2. An immortalized human alveolar type ii epithelial cell line according to claim 1, wherein said line is deposited in China center for type culture collection with a accession number cctccc No. C2022245 at day 8 and 3 of 2022.

3. A method of constructing an immortalized human alveolar type ii epithelial cell line comprising the steps of:

isolated culture of primary human normal alveolar epithelial cells;

constructing recombinant vectors, namely respectively constructing plasmid vectors carrying SV40T antigen, TTF1 gene and c-Myc gene;

gene transfection of primary human alveolar cells three genes were transfected on primary cultured human alveolar epithelial cells;

sub-cell clone screening of human alveolus type II epithelial cells;

identification of immortalized human alveolar type II epithelial cell lines.

4. A method according to claim 3, wherein the recombinant vector in step (2) is selected from a retroviral vector, a lentiviral vector or a CRISPR/CAS plasmid.

5. The method of claim 4, wherein the recombinant plasmid vectors carrying the SV40T antigen, the TTF1 gene and the c-Myc gene are: pRRLsin-SV40T anti-IRES-Puro, pRRLsin-TTF1-IRES-BSD, pRRLsin-c-Myc-IRES-Zeocin.

6. A method according to claim 3, wherein in step (3) the gene transfection of primary human alveolar cells comprises the steps of:

(1) Infecting primary human alveolar epithelial cells with a viral suspension carrying the SV40T antigen gene, culturing, and screening the cells with puromycin;

(2) Infecting the cells obtained in the step (1) by using a virus suspension carrying TTF1 and c-Myc genes, further screening the cells by using blasticidin and bleomycin, cloning the screened cells by subcellular, and selecting a cell clone with an alveolar type II epithelial cell phenotype, namely an immortalized human alveolar type II epithelial cell line.

7. A method for large scale in vitro culture of an immortalized human alveolar type ii epithelial cell line according to any one of claims 1 to 2, comprising the steps of:

cell plane culture: cell lines according to 1-5 x 10 ⁴ /cm ² Inoculating in a cell factory at a density, culturing in a carbon dioxide incubator, and after the cells grow to 90% confluence, adopting trypsin solution for digestion to collect cell suspension;

three-dimensional culture of cell microcarrier, and mixing cell suspension with pre-swelled sterilized microcarrier suspension according to 2-5 x 10 ⁵ Mixing individual cells/ml microcarrier ratio, inoculating into a bioreactor for culture;

continuous collection of cell culture fluid: and after the immortalized human alveolar type II epithelial cells on the microcarriers grow to 35% saturation, starting to collect the cell culture fluid.

8. A lyophilized powder comprising the cell culture fluid of claim 7, which is prepared by cold storage at low temperature, thawing, filtering, and vacuum drying.

9. Use of the lyophilized powder of claim 8 for the preparation of a humanized lung surfactant (PS).

10. A lung surfactant (PS) formulation, characterized in that the lyophilized powder of claim 8 is optionally combined with one or more pharmaceutically acceptable carriers, said formulation comprising an aerosol inhalant, a tracheal drop or an injectable form.

11. Use of a cell line according to claim 1, a lyophilized powder according to claim 8 or a PS formulation according to claim 10 for the manufacture of a medicament for the treatment and/or prevention of a disease associated with a deficiency or dysfunction of a lung surfactant.

12. The use according to claim 11, wherein the pulmonary surfactant deficiency or dysfunction related disease is selected from neonatal respiratory distress syndrome NRDS, secondary respiratory distress syndrome ARDS, and severe pneumonia, meconium aspiration syndrome, new coronavirus pneumonia or chronic obstructive pulmonary disease induced PS deficiency or dysfunction.

13. Use of the lyophilized powder of claim 8 as a pharmaceutical carrier in the preparation of an inhalable medicament.