CN115537403B

CN115537403B - Construction of Endold sensitive cell strain and application thereof in Endold determination

Info

Publication number: CN115537403B
Application number: CN202211532905.4A
Authority: CN
Inventors: 秦玺; 李山虎; 安怡方; 周勇; 裴德宁; 李响; 史新昌; 丁有学; 毕华; 于雷; 黄芳; 朱留强; 王军志
Original assignee: National Institutes for Food and Drug Control
Current assignee: National Institutes for Food and Drug Control
Priority date: 2022-12-02
Filing date: 2022-12-02
Publication date: 2023-03-14
Anticipated expiration: 2042-12-02
Also published as: CN115537403A

Abstract

The invention discloses construction of an Endotoxin sensitive cell strain and application thereof in Endotoxin activity measurement, and provides a method for constructing an Endostatin sensitive cell strain, a method for detecting Endotitin activity and application of UTS2 in construction of the Endostatin sensitive cell strain. The sensitive cell strain and the method provided by the invention can more sensitively, stably and efficiently measure the activity of Endostatin, and have wide application prospects.

Description

Construction of Endolomorphic cell strain and application thereof in Endolomorphic determination

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to construction of an Endothiosensitive cell strain and application thereof in Endothiometry.

Background

Endostatin (Endostatin) is a substance which is separated from the endothelial cell line of mouse and has the function of inhibiting the endothelial cells of blood vessels by applying the genetic engineering technology, is used for treating or retreating the patients with III/IV stage non-small cell lung cancer, and is an auxiliary medicine for the chemotherapy of the cancer patients. The vascular endothelial inhibin is a new biological product for inhibiting angiogenesis, and has the action mechanism of inhibiting the generation of tumor neovascularization by inhibiting the migration of endothelial cells forming blood vessels and blocking the nutrient supply of tumor cells, thereby achieving the purpose of inhibiting the proliferation or the metastasis of tumors.

The Endostatin activity measurement is the measurement of the effective components and content of the medicine and the potency of the medicine, and is an important quality control index for ensuring the effectiveness of the medicine. At present, no effective method aiming at the activity determination of Endostatin exists, and a method capable of stably and efficiently determining the activity of Endostatin is urgently needed in the field.

Disclosure of Invention

In order to make up the defects of the prior art, the invention constructs an Endostatin sensitive cell strain and establishes a more sensitive, stable and efficient Endostatin activity determination method.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a method for constructing an Endostatin sensitive cell line, which comprises knocking down UTS2.

Further, it comprises knocking down UTS2 by RNA interference technology, homologous recombination technology, transcription activation effector nuclease-like technology, zinc finger nuclease technology and CRISPR technology.

Further, UTS2 was knocked down by RNA interference techniques.

Further, the target sequence for knocking down by the RNA interference technology is shown in SEQ ID NO. 3-5.

Further, the target sequence for knocking down by the RNA interference technology is shown as SEQ ID NO. 5.

Further, the RNA interference technology comprises siRNA, shRNA and miRNA.

Further, the RNA interference technology is shRNA.

Further, the Endostatin sensitive cell strain is selected from endothelial cells.

Further, the endothelial cells are selected from vascular endothelial cells.

Further, the vascular endothelial cells include vein endothelial cells and artery endothelial cells.

Further, the vascular endothelial cells are selected from venous endothelial cells.

Further, the vein endothelial cells comprise umbilical vein endothelial cells and cerebral vein endothelial cells.

Further, the venous endothelial cells are selected from umbilical vein endothelial cells.

Further, the umbilical vein endothelial cells include human umbilical vein endothelial cells and non-human animal umbilical vein endothelial cells.

Further, the umbilical vein endothelial cells are selected from human umbilical vein endothelial cells.

The second aspect of the invention provides an Endostatin sensitive cell line, which is constructed by the method of the first aspect of the invention.

In a third aspect, the invention provides a method for detecting Endostatin activity, which comprises using the Endostatin sensitive cell strain of the second aspect to detect.

Further, the method also comprises the steps of adding Endostatin into the Endostatin sensitive cell strain for incubation, and detecting the activity of the Endostatin by a colorimetric method.

Further, the colorimetric method includes an MTT method, an XTT method, and a CCK8 method.

Further, the colorimetric method is a CCK8 method.

Furthermore, the cell density of the Endostatin sensitive cell strain is 5000 cells/mL-11000 cells/mL.

Further, the cell density of the Endostatin-sensitive cell line was 9000 cells/mL.

Further, the method also comprises adjusting the final action concentration of Endostatin to be 0.1mg/ml.

A fourth aspect of the invention provides the use of any one of:

(1) The application of UTS2 in constructing Endostatin sensitive cell strains;

(2) The Endostatin sensitive cell strain of the second aspect of the invention is applied to detecting the activity of Endostatin.

The invention has the advantages and beneficial effects that:

the Endostatin sensitive cell strain and the method for detecting the activity of Endostatin provided by the invention fill the blank of Chinese pharmacopoeia for the method for detecting the activity of the recombinant protein drugs, expand the idea for detecting the activity of the recombinant protein drugs, do not need animal experiments, and have the advantages of simple operation, high sensitivity, reliable result, high accuracy and small variation.

Drawings

FIG. 1 is a graph showing the results of the inhibition of sensitive cells by Endostatin at various concentrations;

FIG. 2 is a graph comparing sensitive cells to primary cells;

FIG. 3 is a graph showing the results of measuring Endostatin activity at different cell densities;

FIG. 4 is a graph showing the results of measuring Endostatin activity at different pre-dilution times.

Detailed Description

The following provides definitions of some terms used in this specification. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The invention provides a method for constructing an Endostatin sensitive cell strain, which comprises knocking down UTS2.

Endostatin is used interchangeably with Endostatin, and the Endostatin may refer to natural Endostatin, recombinant Endostatin, or a functional variant of Endostatin, such as an engineered functional variant, which has one or more amino acid substitutions, deletions, or additions compared to natural Endostatin, and has substantially the same biological functions, such as the activity of inhibiting vascular endothelial cell proliferation, migration, and angiogenesis in vivo. "endostatin" may also refer to a derivative or modification product of native endostatin or a functional variant thereof, such as a polyethylene glycol modification product.

In the present invention, the Endostatin sensitive cell strain is selected from endothelial cells.

The endothelial cells include but are not limited to vascular endothelial cells, renal endothelial cells, lung endothelial cells.

In an alternative embodiment of the invention, the endothelial cells are selected from vascular endothelial cells.

The vascular endothelial cells include but are not limited to venous endothelial cells and arterial endothelial cells.

In another alternative embodiment of the invention, the vascular endothelial cells are selected from venous endothelial cells.

The vein endothelial cells include, but are not limited to, umbilical vein endothelial cells, cerebral vein endothelial cells, and pulmonary vein endothelial cells.

In a preferred embodiment of the invention, the venous endothelial cells are selected from umbilical vein endothelial cells.

The umbilical vein endothelial cells comprise human umbilical vein endothelial cells and non-human animal umbilical vein endothelial cells.

In the present invention, the term "non-human animal" includes all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any domestic or pet animal; and non-mammals, such as chickens, amphibians, reptiles, and the like.

In a particular embodiment of the invention, the umbilical vein endothelial cells are selected from Human Umbilical Vein Endothelial Cells (HUVECs).

In the present invention, UTS2 includes wild type, mutant or fragments thereof. The term encompasses full-length, unprocessed UTS2, as well as any form of UTS2 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of UTS2. UTS2 include human UTS2 as well as UTS2 from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats). As a preferred embodiment, UTS2 is a human gene, gene ID:10911, in the present invention.

In the present invention, the term "knock-down", "knock-out" or "reduction" refers to an experimental method that results in a reduction in the expression of a given gene compared to the expression in a control cell. Knock down of a gene can be accomplished by various experimental means, such as introducing a nucleic acid molecule into a cell, hybridizing it to a portion of the mRNA of the gene that causes its degradation (e.g., shRNA, RNAi, miRNA), or altering the sequence of the gene in a manner that results in reduced transcription, reduced mRNA stability, or reduced mRNA translation.

The invention can use common gene knocking-down/knocking-out/silencing technology to knock-down the UST2, including but not limited to Homologous Recombination (HR) technology, transcription activation effector nuclease-like (TALEN) technology, zinc Finger Nuclease (ZFN) technology, and recently developed regularly clustered spaced short palindromic repeat (CRISPR) technology, RNA interference technology.

Homologous recombination techniques refer to recombination that occurs between sister chromatids (sister chromatins) or between or within DNA molecules containing homologous sequences on the same chromosome. In the gene knocking-down/knocking-out/silencing process, a recombinant vector with the same segment needs to be designed aiming at specific segments at two ends of a target gene, and after the recombinant vector is introduced into an embryonic stem cell, homologous recombination can occur between an exogenous recombinant vector and the same segment in the embryonic stem cell.

Transcription activation effector-like nucleases (TALENs) are endonucleases TALENs which can be constructed to cut specific DNA sequences by coupling TALEs which recognize specific DNA sequences with endonucleases FokI. And FokI needs to form a 2-polymer to exert activity, so that the random shearing probability is greatly reduced. In practical operation, two adjacent target sequences (generally 16-20 bases) with the interval of 13-22 bases are selected at the coding region of a target gene or at the boundary of an exon and an intron and are respectively constructed by TAL recognition modules. And (3) fusing and cloning the two adjacent target recognition modules to the N-terminal of FokI (respectively) to form a eukaryotic expression vector, so as to obtain a TALEN plasmid pair. Co-transferring the TALEN plasmid pair into a cell can achieve target gene knock-out/knockdown. After the TALEN plasmid pair is co-transferred into a cell, the expressed fusion proteins are respectively specifically combined with target sites, because FokI in the two TALEN fusion proteins is close to each other, a dimer is formed, non-specific endonuclease activity is exerted, DNA is cut between the two target sites, and DSB (Double-Strand Breaks) is formed, and a DNA damage repair mechanism is induced. Cells can repair DNA through NHEJ (Non-homologus End Joining), in the process of repair, a certain number of bases are more or less deleted or inserted, so that frame shift is caused, and a target gene knockout/knock-down mutant is formed.

Zinc Finger Nucleases (ZFNs), also known as zinc finger protein nucleases, consist of a DNA recognition domain and a non-specific endonuclease. The DNA recognition domain is composed of a series of Cys2-His2 Zinc finger proteins (Zinc-fingers) in tandem (typically 3-4), each of which recognizes and binds to a specific triplet base. Zinc finger proteins are derived from a family of transcriptional regulators, and are widely present in eukaryotes from yeast to humans, forming an alpha-beta-beta secondary structure. The alpha helix of a single zinc finger is inserted into the major groove of the DNA double helix and specifically recognizes and binds to 3 contiguous bases on the DNA sequence. ZFNs are gene scissors with a localization system, zinc finger proteins for recognition and binding to specific gene sequences, fokI endonucleases can specifically cleave genes of interest by dimerization, and can cleave arbitrary recognition sequences of eukaryotic genomes.

CRISPR is an acquired immune system derived from prokaryotes, the complex that performs the interference function consisting of the proteins Cas and CRISPR-RNA (crRNA). Three types of the system have been found, wherein the second type of the Cas9 system is simple in composition and has been actively applied to the field of genetic engineering. Cas9 targeted cleavage of DNA is achieved by the principle of complementary recognition of two small RNAs, crRNA (CRISPRRNA) and tracrRNA (trans-activating crRNA), to the target sequence. Two small RNAs have now been fused into one RNA strand, sgRNA (single guiderna), capable of recognizing specific gene sequences and guiding Cas9 protein cleavage.

RNA interference technology is a cell reaction process for leading double-stranded RNA into cells to cause the degradation of specific gene RNA, and relates to the joint participation of a plurality of proteins. After entering into the cell, the double-stranded RNA is cleaved into small RNA fragments of only 21-23nt by ribozyme, namely siRNA, which is then combined with RNA-induced silencing complex (RISC) in cytoplasm and is uncoiled into single strand. The sense strand is degraded and the remaining antisense strand directs the RISC to bind to the corresponding complementary mRNA, causing the RISC to cleave and degrade this segment of mRNA, resulting in its inability to translate into proteins or regulate gene expression.

In an embodiment of the invention, UTS2 is knocked down using RNA interference techniques.

Classes of RNA interference include, but are not limited to, siRNA (Short/small interfering RNAs), microRNA (miRNA, microRNA), shRNA (RNA-Short hairpin RNAs).

siRNA refers to ribonucleic acid (RNA) or RNA analogs comprising about 19 to 50 nucleotides (or nucleotide analogs) capable of directing or mediating RNA interference pathways. These molecules may vary in length and may contain varying degrees of complementarity to a target messenger RNA (mRNA) in their antisense strand. The term "siRNA" includes duplexes of two separate strands (i.e., double-stranded RNA) as well as single strands that can form a hairpin structure consisting of a duplex region. The siRNA may be about 19 to 50 nucleotides, or about 25 to 50 nucleotides, or about 30 to 50 nucleotides, or about 35 to 50 nucleotides, or about 40 to 50 nucleotides in length.

micrornas (mirnas, micrornas) generally refer to single-stranded molecules, but in particular embodiments, they may also encompass regions or other strands that are either another region of the same single-stranded molecule or that are partially (10% to 50% complementary chain length), substantially (greater than 50% but less than 100% complementary chain length) or fully complementary to another nucleic acid portion. Thus, a nucleic acid may encompass such molecules: it comprises one or more complementary strands or self-complementary strands, or "complements" of specific sequences comprised by the molecule.

shRNA refers to a single molecule of RNA capable of RNAi and containing a follower strand, loop, and guide strand. The follower strand and the guide strand may be substantially complementary to each other. The term "shRNA" may also include nucleic acids containing moieties other than ribonucleotide moieties, including but not limited to: modified nucleotides, modified internucleotide linkages, non-nucleotides, deoxynucleotides, and nucleotide analogs.

In a specific embodiment of the invention, the expression of UTS2 is inhibited using shRNA.

In the present invention, "target sequence" or "target site" refers to a genomic nucleic acid sequence that defines the portion of the nucleic acid that can specifically bind to a binding molecule under conditions sufficient for binding to occur.

The knockdown target sequence of UTS2 is shown in SEQ ID NO. 3-5.

The knockdown target sequence of UTS2 is shown as SEQ ID NO. 5.

The invention provides a method for detecting Endostatin activity, which comprises the step of detecting by using the Endostatin sensitive cell strain.

In the present invention, the method for detecting the Endostatin activity includes, but is not limited to, a staining counting method, a clone (colony) forming method, and a colorimetric method.

In an embodiment of the invention, the method for detecting the Endostatin activity is a colorimetric method.

The colorimetric methods include, but are not limited to, MTT method, XTT method, CCK8 method, alamar Blue method, lactate Dehydrogenase (LDH) release method, SRB method, ATP assay.

The MTT method is also called MTT colorimetric method. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystalline Formazan (Formazan) and deposit the Formazan in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and an enzyme linked immunosorbent assay detector is used for measuring the light absorption value of the formazan at the wavelength of 570nm, so that the quantity of living cells can be indirectly reflected. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number. The method is widely used for activity detection of some bioactive factors, large-scale screening of anti-tumor drugs, cytotoxicity test, tumor radiosensitivity determination and the like.

XTT is a heterogeneous tetrazolium nitrogen derivative similar to MTT and is reduced to a water-soluble brown ribs and ribs by living cell mitochondrial dehydrogenases, and when XTT is used in combination with an electron coupler, the amount of formation in the ribs and ribs of the nails is positively correlated with the extent of cell proliferation.

The CCK8 method (Cell Counting Kit-8) is a WST-8-based detection reagent widely used for Cell proliferation and cytotoxicity. WST-8 (chemical name: 2- (2-Methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt), a compound similar to MTT, was reduced by a dehydrogenase in mitochondria to the highly water-soluble orange formazan product (formazan) in the presence of the electron carrier 1-Methoxy-5-methylphenazinium dimethylsulfate (1-Methoxy PMS). The more rapid the cell proliferation, the darker the color; the more cytotoxic, the lighter the color, and for the same cells, the shade of the color is proportional to the number of living cells, so cell proliferation and toxicity analysis can be directly performed by using this characteristic.

The main component of Alamar Blue is a redox indicator which exhibits violet Blue non-fluorescence in the oxidized state and converts to a reduced product which fluoresces pink or red in the reduced state, with an absorption peak of 530-560nm and a scattering peak of 590nm.

SRB is a protein-binding dye that binds to basic amino acids in biological macromolecules and changes color in direct proportion to proteins in living cells.

In a particular embodiment of the invention, the activity of Endostatin cells is measured using the CCK8 method.

The specific method comprises the following steps: 1) And (3) cell culture: the ECM culture of Endostatin-sensitive cells (i.e., sensitive HUVEC cells) and primary HUVEC cells supplemented with fetal bovine serum, ECGS, P/O Solution was based on 5% CO at 37 ℃% ₂ The cells are cultured in the incubator until the cells are in a good state and enter a logarithmic growth phase for inoculation.

2) Adding medicine: endostatin was serially diluted 2-fold with buffer to 8 concentration gradients at 0.1mg/mL starting concentration, with two duplicate wells in parallel per concentration gradient. 40 μ L of drug per well was added to a 96-well plate.

3) Inoculation: the cells were digested with 0.25% trypsin, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, resuspended in medium, and viable cells were counted microscopically using a hemocytometer. Adjusting the cell density to 9000/mL, adding 160. Mu.L of cell suspension per well in the preceding 96-well plate, setting at 37 ℃,5% CO ₂ The culture was carried out in an incubator for 96 hours.

4) And (3) detection: using CCK8 assay, adding 22. Mu.L of CCK8 per well, placing at 37 ℃,5% ₂ After 3 hours of incubation in the incubator, the OD was measured at a wavelength of 450nm using a microplate reader.

The invention is further illustrated below with reference to specific examples. It should be understood that the particular embodiments described herein are presented by way of example and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention.

Examples

1. Materials and methods

1.1 Experimental Material

HUVEC primary cells: reserving in a laboratory; endostatin finished product: reserving in a recombined medicine room of the Chinese institute for testing food and drugs; PCDH-flag plasmid: storing in military institute; an SV40 fragment: c, checking for hospital and storing; HEK293T cells: storing in military hospital; pSPAX2, pMD2G: coordinating blood donations; CRISPRV2-A, CRISPRV2-B: purchased from addgene; lipofectamine3000: from invitrogene L3000015; polybrene: purchased from santa cruz; puromycin: purchased from Solarbio; pleti-CRISPRV2: military hospital retention; JLVU6-GFP-Kan-Puro: purchased from Jinshiu.

Experimental reagent

PEI transfection reagent: 50 XTAE buffer (Bio-Industrial, B548101, china), cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, CK04-1000T, japan), trypan blue dye solution (15250-061, gibco, USA), PBS, pH 7.2 (20012027, gibco, USA), 0.25% trypsin solution (25200-056, gibco, USA).

293T growth Medium: DMEM (GIBCO, C11995500 BT) +10% fetal bovine serum (GIBCO, # 10099) +1% double antibody (GIBCO, # 15240).

HUVEC growth medium: ECM (SclenCell, # 1001) +5% fetal bovine serum (SclenCell, # 0025) +1% ECGS (SclenCell, # 1052) +1% P/OSolution (SclenCell, # 0503) +1% diabody (GIBCO, # 15240).

96-well plate: CORNING,3635.

CCK8：DOJINDO，CK04。

Laboratory apparatus

PCR instruments (PTC-200, MJ research, USA); an electrophoresis apparatus (PowerPac 300, bio-Rad, USA); gel imagers (5200 Multi, tanon, china); 96-well cell plate (3903, costar, USA); high speed centrifuges (5424, eppendorf, USA); high speed refrigerated centrifuge (Auegr X22, beckman, USA); high speed cryogenic centrifuges (5417R, eppendorf, USA); superclean bench (JJT-1300, beijing Otsu Tan, china); data analysis software; softMax Pro software; graphPad Prism software.

1.2 Experimental methods

1.2.1 construction of immortalized HUVEC cells

1) A PCDH-flag-SV40 plasmid was constructed, and the primers are shown in Table 1.

PCR amplifying SV40 segment and connecting with carrier, coating plate after conversion, selecting bacterial colony monoclonal PCR identification and sequencing, screening bacterial liquid with correct sequence, proceeding plasmid amplification.

2) Construction of SV 40-expressing HUVEC cells

(1) Packaging lentivirus with 293T cells (packaging plasmid: pSPAX 2.25 μ L, pMD2G 0.75 μ L; target plasmid: P is selected from CDH-flag-SV40 μ L), adding three plasmids into 300 μ L DMEM basal medium, mixing and standing for 5min, adding 6 μ L10 XPEI into 300 μ L DMEM basal medium, mixing and standing for 5min, mixing and standing for 25min, dripping into 293T cells, changing 8ml ECM medium after 10-12h, collecting supernatant 24hr after changing, storing in a 4 deg.C refrigerator, adding 8ml ECM medium into cells, collecting supernatant 24hr after, mixing supernatant of the previous day, filtering with 0.45 μ M filter, subpackaging, and storing in a-80 deg.C refrigerator.

(2) HUVEC were passaged one day before infection of HUVEC cells with lentivirus to reach a density of 30% -40% when infected with virus and 30min before infection, ECM medium containing polybrene 10. Mu.g/mL was added to each HUVEC dish and placed in CO ₂ Culturing in an incubator, adding 4mL of lentivirus into each dish of HUVEC after 30min, replacing fresh ECM culture medium 24hr after infection, transferring HUVEC (named HUVEC-Flag-SV 40) infected with lentivirus to 10 generations because the cells have no resistance after infection and the HUVEC cells die after 10 generations under the condition of no large T antigen (SV 40), obtaining the HUVEC cell line which stably expresses the large T antigen after survival, and detecting whether to express SV40 protein (see whether to express Flag) by using Western Blot.

1.2.2 Screening for sensitive genes

1) Amplification of lentivirus libraries

Firstly, 293T cells are subjected to amplification culture, and packaged by lentiviruses, namely CRISPRV2-A and CRISPRV2-B, and transfected into the 293T cells.

Respectively mixing uniformly for 5min, slowly dripping B into A, gently blowing uniformly, standing for 25min, dripping into a culture dish, shaking uniformly, placing into an incubator at 37 ℃ for 8 h, discarding the culture solution containing the transfection reagent, replacing with fresh culture, collecting the culture solution containing virus respectively after 24h and 48 h, mixing the collected virus solutions, filtering with a 0.45 mu M filter, subpackaging, and storing in a refrigerator at-80 ℃.

2) Infecting HUVEC-flag-SV40 with lentivirus, and screening Endostatin resistance gene by CRISPR library

(1) The required virus volume was 66.7 μ L at MOI = 0.4.

(2) When the total number of cells cultured>3.3×10 ⁸ In this case, the appropriate volume of cell sap was inoculated, and 3X 10 cells were initially inoculated per flask ⁶ Is smallCells, a total of 75 flasks were inoculated, and the total cell count was about 2.25X 10 ⁸ One cell (2 ten thousand genes, 6 targets per gene, 250-fold coverage, 3 total required with MOI = 0.4). (3 additional flasks, 2 of which were not virus infected as control cells and the other flask was used for counting to determine whether the MOI was 0.4 for this experiment).

(3) The next day, when the density of the cells after adherence is about 40%, the culture solution is sucked off, 10mL of ECM culture medium containing polybrene with the concentration of 10 μ g/mL is added into each culture bottle, and CO is added ₂ After 30min of incubation, 10Ml of ECM culture medium and 2 Ml of virus solution were added to each flask.

(4) Lentiviral infection was followed 24hr later by removal of lentivirus, replacement with 20mL of fresh ECM medium, and addition of 10mL of ECM medium containing puromycin at a final concentration of 2. Mu.g/mL.

(5) When the control group of uninfected lentiviruses are screened by puromycin to be dead, a bottle of control group cells and virus-infected cells are taken, counted by a counting gun, and an MOI value is calculated. The surviving cells were those with the sgRNA library that were successfully infected. The remaining lentivirus-infected HUVEC-flag-SV40 was replaced with 30ml of fresh ECM medium.

(6) Surviving cells were resistance-screened for 3 days by adding ECM medium containing puromycin at a final concentration of 2. Mu.g/ml, and the cells were divided into 3 groups of 6.0X 10 cells based on cell count ⁷ I.e. the library sgRNA targets cover 500 times. Wherein the cell group is named HUVEC P0 as day0 cell group and used as sgRNA sequencing reference line, centrifuging at 1500 rpm for 5min, discarding supernatant, resuspending and washing with PBS twice, discarding supernatant, directly storing in-80 deg.C refrigerator, inoculating the rest two groups of cells at 40 cells of 175 mm ² In each culture bottle, 20 bottles are divided into a drug-adding screening group and a blank control group, and the initial inoculation cell number of each culture bottle is 3 multiplied by 10 ⁶ 。

(7) Selecting Endostatin with final concentration of 100 μ g/mL, culturing without drug-adding group and drug-adding group, waiting for cell proliferation of drug-adding group and control group to be less than or equal to 1.2 × 10 ⁸ Subcultured in time, and the inoculation number of each group is 6.0 multiplied by 10 ⁷ And recording and comparing the proliferation difference of the two groups of cells. Two groups of cells need to be screened for 8-10 generations respectively to ensure the stable enrichment of potential target genes.

(8) Centrifuging two groups of cells for 5min at 1500 rpm, removing supernatant, carrying out heavy suspension and cleaning twice by PBS, removing supernatant, collecting cells, clearly marking 20 mu L of each collected day0 group, endostatin group, blank control group 3 group cells and A and B library plasmids, and sending dry ice to Haichou Caucai Biotechnology Limited for whole genome sequencing to obtain the potential target gene.

3) According to the sequencing result, six potential genes are selected in total, and after the genes are knocked out independently, the knocked-out genes are finally determined to be UTS2 according to the CCK8 experiment result.

1.2.3 construction of Endostatin-sensitive Gene knock-out plasmid

1) Gene knockdown target sequences (Table 3)

2) The target sequence was constructed on JLVU6-GFP-Kan-Puro plasmid.

3) The lentivirus was packaged in HEK293T (as above) and filtered through a 0.45 μ M filter and aliquoted and stored in a freezer at-80 ℃.

4) The cells were infected with HUVEC-flag-SV40 (as described above) and designated HUVEC-UTS 2# (SEQ ID NO. 3), HUVEC-UTS 2# (SEQ ID NO. 4), HUVEC-UTS 2# (SEQ ID NO. 5).

5) CCK8 detects whether UTS2 gene is sensitive to Endostatin after being knocked down.

(1) Cell culture

Culturing primary HUVEC cells and sensitive HUVEC cells in ECM supplemented with fetal bovine serum, ECGS, P/O Solution at 37 deg.C with 5% CO ₂ The cells are cultured in the incubator until the cells are in a good state and enter a logarithmic growth phase for inoculation.

(2) Dosing

The concentration of the finished Endostatin product is 5mg/mL, and the solution is used as an initial concentration to perform 2-fold serial dilution with a buffer solution to form 8 concentration gradients, and each concentration gradient is provided with two parallel multiple holes. 40 μ L of drug per well was added to a 96-well plate.

(3) Inoculation of

The cells were digested with 0.25% trypsin, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, resuspended in medium, and viable cells were counted microscopically using a hemocytometer. Adjusting the cell density to 5000 cells/mL, adding 160. Mu.L of cell suspension per well in the former 96-well plate, setting at 37 ℃,5% CO ₂ The culture was carried out in an incubator for 96 hours.

(4) Detection

Using CCK8 assay, adding 22. Mu.L of CCK8 per well, placing at 37 ℃,5% ₂ After 3 hours of incubation in the incubator, the OD was measured at a wavelength of 450nm using a microplate reader.

1.2.4 Establishment of method for measuring activity of Endostatin sensitive cells

1.2.4.1 determination of Pre-dilution factor

According to the test results, the cell density is 800 cells/hole, the concentration of the finished product is 5mg/mL, the dilution is not carried out, the action time of the medicine is 96h, and the CCK8 detection is carried out.

1.2.4.2 comparison of Linear responses at different cell densities

According to the previous test results, the concentration of Endostatin pre-dilution is 1mg/mL, the action time of the drug is 96h, and the detected cell density is 5000, 7000, 9000, 11000 and 13000 dose-response curves respectively.

1.2.4.3 Linear reaction at different pre-dilution times

According to the previous test results, the cell density is 9,000/mL, the final concentration of Endostatin is adjusted to 0.1 mg/L, 0.3 mg/L, 0.4 mg/L and 0.5 mg/L, the dilution factor is2 times, and the dose-response curves of different initial concentrations are detected.

1.2.5 methodological validation

1.2.5.1 accuracy

Endostatin samples were assayed for activity by the newly established method, and 75%, 100%, 125% and 150% solutions at the mass concentration of the pre-diluent (1 mg/mL) were prepared, two wells for each dose, and three plates were repeated for four consecutive days.

1.2.5.2 precision

The activity of Endostatin samples is determined by a newly-built method, 75%, 100%, 125% and 150% solutions with the mass concentration of the pre-diluent (1 mg/mL) are prepared, each dose is provided with two duplicate wells, three plates are repeatedly made, and the steps are continuously repeated for four days.

2. Results of the experiment

2.1 Sensitive cell strain

CCK8 detection results show that immortalized HUVEC cells have higher sensitivity to Endostatin after knocking down UTS2 (figure 1), wherein HUVEC-UTS2-KO3# has the best sensitivity to Endostatin (figure 2).

2.2 Establishment of detection method

Parameters such as drug pre-dilution, cell plating density and the like are optimized respectively. By comparing ED ₅₀ Values and signal-to-noise ratios, the detection scheme was initially determined: the cell plating density was 9,000 cells/mL (FIG. 3), the final concentration of Endostatin drug was 0.1mg/mL, 2-fold dilution, and the drug stimulation time was 96 hours (FIG. 4).

2.3 Establishment of detection method

2.3.1 accuracy

As shown in Table 4, the recovery rates of the 75%, 100%, 125% and 150% solutions were (103.97. + -. 11.47)%, (99.84. + -. 8.70)%, (94.36. + -. 7.31)%, (103.97. + -. 12.30)%, respectively, and the RSD values were less than 15%, indicating that the method had good accuracy.

2.3.2 precision

As shown in Table 5, RSD values between the average relative biological activity values obtained by repeating the above four days were 2.12% to 17.70%, indicating good precision.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims

1. A method for constructing an Endostatin sensitive cell strain is characterized in that UTS2 is knocked down, and the knocked-down target sequence of UTS2 is shown as SEQ ID NO. 3-5.

2. The method of claim 1, wherein the Endostatin-sensitive cell strain is selected from the group consisting of endothelial cells.

3. An Endostatin-sensitive cell line constructed by the method of any one of claims 1 to 2.

4. A method for detecting the activity of Endostatin, which comprises using the Endostatin sensitive cell line of claim 3.

5. The method of claim 4 which comprises adding Endostatin to the Endostatin sensitive cell line and incubating, and measuring the activity of Endostatin by colorimetry.

6. The method of claim 5, wherein the cell density of the Endostatin-sensitive cell line is 5000 cells/mL-11000 cells/mL.

7. Any one of the following applications:

(1) The UTS2 is applied to the construction of Endostatin sensitive cell strains, wherein the UTS2 is a knocked-down UTS2, and a knocking-down target sequence of the UTS2 is shown as SEQ ID NO. 3-5;

(2) Use of the Endostatin sensitive cell line of claim 3 to detect the activity of Endostatin.