CN108251378A - A kind of interstital stem cell excretion body for being overexpressed PTGDS genes and its preparation method and application - Google Patents
A kind of interstital stem cell excretion body for being overexpressed PTGDS genes and its preparation method and application Download PDFInfo
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- CN108251378A CN108251378A CN201810060438.7A CN201810060438A CN108251378A CN 108251378 A CN108251378 A CN 108251378A CN 201810060438 A CN201810060438 A CN 201810060438A CN 108251378 A CN108251378 A CN 108251378A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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Abstract
The present invention relates to a kind of interstital stem cell excretion bodies for being overexpressed PTGDS genes and its preparation method and application, belong to gastric cancer medicament preparing technical field.The interstital stem cell excretion body provided by the invention for being overexpressed PTGDS genes is after being overexpressed Adenovirus Transfection interstital stem cell using PTGDS, to be obtained after cultivating, isolating and purifying.The interstital stem cell excretion body efficient target stomach cancer cell provided by the invention for being overexpressed PTGDS genes inhibits the growth and migration of stomach cancer cell, and then inhibits tumour growth.
Description
Technical field
The present invention relates to gastric cancer medicament preparing technical fields, and in particular to a kind of interstitial for being overexpressed PTGDS genes is done carefully
It is extracellular to secrete body and its preparation method and application.
Background technology
Gastric cancer is the fifth-largest common malignant tumour in the world, and the death rate occupies third position, and in China, gastric cancer height
The third position of most common cancer is occupied, has been second largest Cancer death reason.Clinical treatment gastric cancer medicament mainly has 5- fluorine at present
The chemotherapeutics such as uracil, these drugs can significantly inhibit the progress of tumour, but since it is to the special of tumor tissues
Property is poor, and has very strong side effect.Therefore, we urgently need one kind not only with anti-gastric cancer cell growth but also with targeted therapy
" biological agent " of effect.
PGD2 is one kind of prostaglandin, is important cell signaling molecule, its synthesis mainly passes through following biology
Process：Phospholipase A on cell membrane is converted to arachidonic acid, and the latter is in the catalysis of Cycloxygenase (including COX-1 and COX-2)
Under, unstable intermediate product prostaglandin H2 is broken down into, prostaglandin H2 is again by three kinds of different enzymes：Prostaglandin D is closed
Into enzyme (PGDS), Prostaglandin E Synthase and prostaglandin F synzyme are converted into more stable PGD2, PGE2, PGF2.PGDS
The crucial enzyme system of prostaglandin of series, and synthesized including lipocalin-type PGD synzyme (L-PTGDS) and hematopoiesis type PGD
Enzyme (H-PTGDS).Although existing report discloses and PTGDS is overexpressed in tumour cell can to efficiently control external gastric cancer thin
The growth of born of the same parents has the potentiality (number of patent application for the treatment of gastric cancer：201611203449.3), but the prior art still lacks practical energy
The drug of enough efficient targeted therapy gastric cancers.
Invention content
The purpose of the present invention is to provide a kind of interstital stem cell excretion bodies for being overexpressed PTGDS genes and preparation method thereof
And application.The interstital stem cell excretion body efficient target stomach cancer cell provided by the invention for being overexpressed PTGDS genes, inhibits gastric cancer
The growth and migration of cell, and then inhibit tumour growth.
The present invention provides a kind of interstital stem cell excretion body for being overexpressed PTGDS genes, the overexpression PTGDS genes
The preparation method of interstital stem cell excretion body include the following steps：
1) PTGDS is overexpressed Adenovirus Transfection interstital stem cell, obtaining PTGDS through screening is overexpressed interstital stem cell；
2) PTGDS that the step 1) obtains is overexpressed interstital stem cell and is seeded to the low sugar DMEM trainings containing fetal calf serum
It supports in base and carries out the first amplification cultivation, when cell fusion is to 70~80%, the second amplification is carried out using serum free medium and is trained
It supports, supernatant is collected after 48h, centrifugation removal floating living cells obtains the supernatant of the body containing excretion；
3) supernatant of the body containing excretion that the step 2) obtains is isolated and purified using sucrose density gradient centrifugation,
Obtain being overexpressed the interstital stem cell excretion body of PTGDS genes.
In the present invention, in the step 1), PTGDS is overexpressed adenovirus with 108Titre transfected.
In the present invention, containing the tire that mass concentration is 10% in the low sugar DMEM culture mediums containing fetal calf serum described in step 2)
Cow's serum.
Preferably, the condition of the step 2) centrifugation centrifuges 10min for 300 × g.
Preferably, described isolate and purify of step 3) includes the following steps：
A) supernatant of body containing excretion is centrifuged into 10min in 2000 × g, obtains the supernatant of removal cell fragment；
B) supernatant of the removal cell fragment is centrifuged into 30min in 10000 × g, obtains the supernatant of removal organelle；
C) supernatant of removal organelle that step b) is obtained is subjected to the first concentration, obtains the first concentrate；
D) the first concentrate that step c) is obtained moves to sucrose/heavy water density pad of 1/4 times of volume, 100000 × g from
Heart 3h collects sucrose/heavy water layer of bottom, carries out the second concentration, obtains the second concentrate；
E) the second obtained concentrates of the step d) are washed, obtains being overexpressed PTGDS genes after filtration sterilization
Interstital stem cell excretion body.
Preferably, it is the conditional sampling of first concentration and the described second concentration：1000 × g centrifuges 30min.
Preferably, the molecular cut off of first concentration and the described second concentration is 100kDa.
Preferably, the step d) washings are carried out using PBS buffer solution, and the number of the washing is 3 times.
The present invention also provides it is a kind of be overexpressed PTGDS genes interstital stem cell excretion body preparation method, including with
Lower step：
1) PTGDS is overexpressed Adenovirus Transfection interstital stem cell, obtaining PTGDS through screening is overexpressed interstital stem cell；
2) PTGDS that the step 1) obtains is overexpressed interstital stem cell and is seeded to the low sugar DMEM trainings containing fetal calf serum
It supports in base and carries out the first amplification cultivation, when cell fusion is to 70~80%, the second amplification is carried out using serum free medium and is trained
It supports, supernatant is collected after 48h, centrifugation removal floating living cells obtains the supernatant of body containing excretion；
3) supernatant of body containing excretion that the step 2) obtains is isolated and purified using sucrose density gradient centrifugation, obtained
To the interstital stem cell excretion body for being overexpressed PTGDS genes
The present invention also provides the interstital stem cell excretion body of PTGDS genes or above-mentioned is overexpressed described in above-mentioned technical proposal
The interstital stem cell excretion body of overexpression PTGDS genes that preparation method described in technical solution obtains is preparing prevention or treatment stomach
Application in cancer drug.
The present invention provides a kind of interstital stem cell excretion bodies for being overexpressed PTGDS genes.Overexpression provided by the invention
The interstital stem cell excretion body of PTGDS genes can target tumor or damage location, PTGDS can specificity inhibit gastric cancer it is thin
Born of the same parents, the interstital stem cell excretion body provided by the invention for being overexpressed PTGDS genes can efficient target stomach cancer cell, inhibition gastric cancer
The growth and migration of cell, and then inhibit tumour growth.Result of the test shows overexpression PTGDS genes provided by the invention
Interstital stem cell excretion body targeting is high, has no toxic side effect, and can be good at controlling gastric cancer progress.
Description of the drawings
(virus transfection is imitated for interstital stem cell form and GFP after the Adenovirus Transfection that Fig. 1 is provided for the embodiment of the present invention 1
The labelled protein of rate) fluorescence photo (400 times)；
Fig. 2 is the interstital stem cell excretion body for the overexpression PTGDS genes that the embodiment of the present invention 1 provides and the unloaded gland of control
The separation qualification result figure of the excretion body of virus transfection；
Fig. 3 is the quantitative PCR inspection for the interstital stem cell excretion body of overexpression PTGDS genes that the embodiment of the present invention 1 provides
Survey result figure；
Fig. 4 is that the interstital stem cell excretion body of overexpression PTGDS genes that the embodiment of the present invention 2 provides is thin to SGC-7901
The influence result figure of the clonality of born of the same parents；
Fig. 5 is that the interstital stem cell excretion body of overexpression PTGDS genes that the embodiment of the present invention 2 provides is thin to SGC-7901
The influence result figure of the amplification in vitro of born of the same parents；
Fig. 6 is that the interstital stem cell excretion body of overexpression PTGDS genes that the embodiment of the present invention 2 provides is thin to SGC-7901
The influence result figure of the transfer ability of born of the same parents；
Fig. 7 is that the interstital stem cell excretion body of overexpression PTGDS genes that the embodiment of the present invention 3 provides is thin to SGC-7901
The influence result figure of one-tenth knurl ability in cell space；
Fig. 8 is the result figure of the tumor weight that the embodiment of the present invention 3 provides and growth curve；
Fig. 9 be the embodiment of the present invention 3 provide overexpression PTGDS genes interstital stem cell excretion body to knurl tissue shape
The influence result figure of state.
Specific embodiment
The present invention provides a kind of interstital stem cell excretion body for being overexpressed PTGDS genes, the overexpression PTGDS genes
The preparation method of interstital stem cell excretion body include the following steps：
1) PTGDS is overexpressed Adenovirus Transfection interstital stem cell, obtaining PTGDS through screening is overexpressed interstital stem cell；
2) PTGDS that the step 1) obtains is overexpressed interstital stem cell and is seeded to the low sugar DMEM trainings containing fetal calf serum
It supports in base and carries out the first amplification cultivation, when cell fusion is to 70~80%, the second amplification is carried out using serum free medium and is trained
It supports, supernatant is collected after 48h, centrifugation removal floating living cells obtains the supernatant of body containing excretion；
3) supernatant of body containing excretion that the step 2) obtains is isolated and purified using sucrose density gradient centrifugation, obtained
To the interstital stem cell excretion body for being overexpressed PTGDS genes.
The present invention provides a kind of interstital stem cell excretion body for being overexpressed PTGDS genes, the excretions of abbreviation PTGDS modifications
Body, the PTGDS genes are prostaglandin D2 synzyme.
PTGDS is overexpressed Adenovirus Transfection interstital stem cell by the present invention, and obtaining PTGDS overexpression interstitials through screening does carefully
Born of the same parents.The present invention is overexpressed adenovirus source to the PTGDS does not have special restriction, and use is well known to those skilled in the art
PTGDS is overexpressed the conventional commercial product of adenovirus, and the PTGDS mistakes of hundred Bioisystech Co., Ltd of Australia are such as answered purchased from Suzhou
Express adenovirus.The present invention to the source of the interstital stem cell also without special restriction, it is normal using those skilled in the art
The acquisition methods of interstital stem cell are advised, it is such as artificial to obtain or buy.The present invention does not have the method manually obtained yet
Special restriction, using the preparation method of conventional interstital stem cell, such as reference literature Human mesenchymal stem
cells isolated from the umbilical cord (Qiao Chun et al.Human mesenchymal
stem cells isolated from the umbilical cord.Cell Biol Int.2008 Jan；32(1):8-
15) it is prepared by the method involved in.The present invention first cultivates the interstital stem cell of acquisition preferably before transfection, between described
The condition of culture of matter stem cell is preferably 37 DEG C, 5%CO2It is cultivated under the conditions of saturated humidity.In the present invention, between described
Matter stem cell is cultivated to third generation cell, and when cell density reaches 50%, is preferably transfected.
The present invention does not have the method for the transfection special restriction, using virus transfection well known to those skilled in the art
Method is directly added into virus liquid after reaching 50% such as cell density and is transfected.In the present invention, the PTGDS crosses table
Up to adenovirus preferably with 108Titre transfected.Present invention preferably employs fluorescence microscope transfection efficiencies.
In the present invention, the screening technique of the PTGDS overexpressions interstital stem cell is preferably specially：Using GFP as PTGDS
Tag molecule, the positive cell by fluorescence microscope GFP be PTGDS be overexpressed interstital stem cell, in other words,
Pass through the overexpression situation of GFP indirect reactions PTGDS.
After obtaining PTGDS overexpression interstital stem cells, the present invention preferably passes on PTGDS overexpression interstital stem cells
Culture is preserved after more preferably passing on 3~5 times, spare.The present invention does not have the condition of the secondary culture special limit
It is fixed, using trypsin digestion cell secondary culture method well known to those skilled in the art.
After obtaining PTGDS overexpression interstital stem cells, PTGDS overexpression interstital stem cells are seeded to ox containing tire by the present invention
The first amplification cultivation is carried out in the low sugar DMEM culture mediums of serum, when cell fusion is to 70~80%, using free serum culture
Base carries out the second amplification cultivation, and supernatant is collected after 48h, and centrifugation removal floating living cells obtains the supernatant of body containing excretion.In the present invention
In, containing the fetal calf serum that mass concentration is 10% in the low sugar DMEM culture mediums containing fetal calf serum.The present invention is to described low
The composition of sugared DMEM culture mediums is not particularly limited, using conventional commercial low sugar DMEM culture mediums well known to those skilled in the art
.It is of the invention preferred when cell fusion is to 75%, the second amplification cultivation is carried out using serum free medium.In the present invention
In, the condition of the centrifugation removal floating living cells is preferably 300 × g centrifugations 10min.
After obtaining the supernatant of body containing excretion, the present invention is detached using sucrose density gradient centrifugation to containing excretion body supernatant
Purifying obtains being overexpressed the interstital stem cell excretion body of PTGDS genes.The present invention does not have the sucrose density gradient centrifugation
Special restriction, using sucrose density gradient centrifugation well known to those skilled in the art.In the present invention, the separation
Purifying (sucrose density gradient centrifugation) method preferably includes following steps：
A) supernatant of body containing excretion is centrifuged into 10min in 2000 × g, obtains the supernatant of removal cell fragment；
B) supernatant of the removal cell fragment is centrifuged into 30min in 10000 × g, obtains the supernatant of removal organelle；
C) supernatant of removal organelle that step b) is obtained is subjected to the first concentration, obtains the first concentrate；
D) the first concentrate that step c) is obtained moves to sucrose/heavy water density pad of 1/4 times of volume, 100000 × g from
Heart 3h collects sucrose/heavy water layer of bottom, carries out the second concentration, obtains the second concentrate；
E) the second obtained concentrates of the step d) are washed, obtains being overexpressed PTGDS genes after filtration sterilization
Interstital stem cell excretion body.
The present invention will contain excretion body supernatant and centrifuge 10min in 2000 × g, obtain the supernatant of removal cell fragment.In this hair
In bright, the centrifugation preferably carries out under the conditions of 4 DEG C.
After obtaining the supernatant of removal cell fragment, the present invention centrifuges the supernatant for removing cell fragment in 10000 × g
30min obtains the supernatant of removal organelle.In the present invention, the centrifugation preferably carries out under the conditions of 4 DEG C.
After obtaining the supernatant of removal organelle, the supernatant of the removal organelle is carried out the first concentration by the present invention, is obtained
First concentrate.In the present invention, the molecular cut off of first concentration is preferably 100 kDa, and first concentration is preferred
30min is centrifuged in 1000 × g.
After obtaining the first concentrate, first concentrate is moved to sucrose/heavy water density of 1/4 times of volume by the present invention
Pad, 100000 × g centrifugation 3h collect sucrose/heavy water layer of bottom, carry out the second concentration, obtain the second concentrate.In the present invention
In, the concentration of sucrose is preferably 30% in the sucrose/heavy water density pad, and density is preferably ρ=1.210g/cm3.In the present invention
In, the centrifugation preferably carries out under the conditions of 4 DEG C.In the present invention, the molecular cut off of second concentration is preferably
100kDa, second concentration preferably centrifuge 30min in 1000 × g.In the present invention, it before second concentration, preferably incites somebody to action
To sucrose/heavy water layer be diluted, the dilution is preferably PBS buffer solution with solvent, is preferably diluted to without apparent precipitation,
Liquid is to transparent.In the present invention, the centrifugation preferably carries out under the conditions of 4 DEG C.
After obtaining the second concentrate, the present invention washs second concentrate, is overexpressed after filtration sterilization
The interstital stem cell excretion body of PTGDS genes.In the present invention, the washing is preferably carried out using PBS buffer solution, the washing
Number be preferably 3 times.In the present invention, the filtration sterilization is preferably carried out using sterilised membrane filter, the hole of the sterilised membrane filter
Diameter is preferably 0.22 μm.
In the present invention, the interstital stem cell excretion body for being overexpressed PTGDS genes carries out preferably under the conditions of -70 DEG C
It preserves.It obtains after being overexpressed the interstital stem cell excretion body of PTGDS genes, the present invention is between the overexpression PTGDS genes
The quantification of protein detection method of matter stem cell excretion body does not have special restriction, using albumen well known to those skilled in the art
Quality detection kit, such as BCA quantification of protein kits.
The present invention also provides it is a kind of be overexpressed PTGDS genes interstital stem cell excretion body preparation method, including with
Lower step：
1) PTGDS is overexpressed Adenovirus Transfection interstital stem cell, obtaining PTGDS through screening is overexpressed interstital stem cell；
2) PTGDS that the step 1) obtains is overexpressed interstital stem cell and is seeded to the low sugar DMEM trainings containing fetal calf serum
It supports in base and carries out the first amplification cultivation, when cell fusion is to 70~80%, the second amplification is carried out using serum free medium and is trained
It supports, supernatant is collected after 48h, centrifugation removal floating living cells obtains the supernatant of body containing excretion；
3) supernatant of body containing excretion that the step 2) obtains is isolated and purified using sucrose density gradient centrifugation, obtained
To the interstital stem cell excretion body for being overexpressed PTGDS genes.
The detailed preparation parameter that the present invention is overexpressed the preparation method of the interstital stem cell excretion body of PTGDS genes is for example above-mentioned
Described in content, details are not described herein.
The present invention also provides the interstital stem cell excretion body of PTGDS genes or above-mentioned is overexpressed described in above-mentioned technical proposal
The interstital stem cell excretion body of overexpression PTGDS genes that preparation method described in technical solution obtains is preparing prevention or treatment stomach
Application in cancer drug.In the present invention, the dosage form of the drug is preferably liquid preparation.
With reference to specific embodiment to a kind of interstital stem cell excretion body for being overexpressed PTGDS genes of the present invention
And its preparation method and application be further described in detail, technical scheme of the present invention includes but not limited to following embodiment.
Embodiment 1
Used main material and source difference are as follows：
MSC cultivate reagent low sugar DMEM, fetal calf serum (Gibco Products), trypsase (Sigma Products),
Carbon dioxide incubator (Forma companies), serum free medium (Shanghai Yi Kesai companies；
Inverted microscope, fluorescence microscope, biomicroscope, electron microscope, superclean bench, desk centrifuge；
PTGDS is overexpressed adenovirus vector (answering hundred Bioisystech Co., Ltd of Australia in Suzhou), heavy water (D2O, upper SeaBird match are public
Department), analyze pure sucrose (Guangzhou Chemical Reagent Factory), rabbit-anti people CD9 antibody (Bioworld Technology companies of the U.S.), rabbit
Anti-human CD63 antibody (Epitomics companies of the U.S.), the mountain that BCA protein quantifications kit, horseradish peroxidase (HRP) mark
Goat anti-rabbit igg secondary antibody (Beijing health be ShiJi Co., Ltd), HRP chemiluminescent substrates, 100-kDaMWCO ultra-filtration centrifuge tubes, 0.22 μm
Sterilised membrane filter (Millipore companies of the U.S.)；Transmission electron microscope (FEITecnai12, Philips company)；Quantitative PCR tries
Agent (Beijing Cwbio companies).
It is overexpressed the acquisition of the interstital stem cell excretion body (the excretion body of PTGDS modifications) of PTGDS genes
(1) by interstital stem cell in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, cultivates successful interstital stem cell
Form is shown in Fig. 1.
(2) PTGDS is overexpressed adenovirus and its comparison virus with 108Titre carry out interstital stem cell transfection, lead to
The transfection efficiency of fluorescence microscope interstital stem cell is crossed, Fig. 1 is the interstital stem cell form and GFP after Adenovirus Transfection
The fluorescence photo (400 times) of (labelled protein of virus transfection efficiency).With reference to white light photo, transfection efficiency (figure more than 80%
1)。
(3) growth selection 3~5 generation PTGDS in good condition are overexpressed interstital stem cell and first use containing 10% fetal calf serum
Low sugar DMEM medium cultures are changed serum free medium culture when cell fusion is to 70%~80%, are collected in culture after 48h
Clearly, 300 × g centrifuge 10min to remove the living cells of floating, for be overexpressed the interstital stem cell excretion body of PTGDS genes (with
It is lower with PTGDS modification excretion body surface show) separation.
(4) the interstital stem cell supernatant of collection is centrifuged into 10min in 4 DEG C, 2000 × g, to remove cell fragment；It collects
Supernatant is after 4 DEG C, 10000 × g centrifugation 30min, to remove organelle；Supernatant is transferred to 100-kDaMWCO ultra-filtration centrifuge tubes,
4 DEG C, 1000 × g centrifugation 30min concentrated；Concentrate is slowly moved to 5ml30% sucrose/heavy water density pad (ρ=
1.210g/cm3), 4 DEG C, 100000 × g centrifugations 3h；Bottom 5ml sucrose/heavy water layer (containing exosome) is collected, is added after PBS dilutions
Enter in 100-kDaMWCO ultra-filtration centrifuge tubes, 4 DEG C, 1000 × g centrifugation 30min, PBS wash 3 times；Finally with 0.22 μm of sterile filter
Membrane filtration degerming dispenses after -70 DEG C of preservations, and carries out quantification of protein detection with BCA quantification of protein RNA isolation kit.
(5) grown form of transmission electron microscope observing excretion body：The excretion body of PTGDS modifications and the unloaded Adenovirus Transfection of control
Each 20 μ L of excretion body (following with control excretion body surface show), dropwise addition is on the load sample copper mesh of diameter 2mm after abundant mixing, in room
After temperature stands 5min, copper mesh edge residual liquid is gently sucked with filter paper, then copper mesh is buckled in 30g/L phosphotungstic acids
(pH6.8) on drop, negative staining 5min, finally dries copper mesh under incandescent lamp at room temperature, is placed under transmission electron microscope and observes and clap
Excretion body according to, PTGDS modification and the separation qualification result of excretion body is compareed as shown in Fig. 2, wherein, Tu2AWei：Transmission electron microscope
Observe its form (scale 100nm) result figure.As shown in Figure 2 A, the excretion body by PTGDS modifications and control excretion body are straight
Diameter is about 100nm capsule balloon-shaped structures；
(6) Westernblot detects the excretion body of PTGDS modifications and the surface markers albumen of control excretion body：It prepares
15%SDS-PAGE running gels add in 1/4 after fully cracking the PTGDS of the said extracted excretion bodies modified and control excretion body
5 × SDS sample-loading buffers of volume, boil 5min, and by 200 μ g protein total amount loadings, electrotransfer (350mA, 120min) will
Protein is gone on pvdf membrane, and 1h is closed with the TBS/T room temperatures of the skim milk containing 50g/L, respectively with rabbit-anti people CD9 antibody and rabbit
Anti-human CD81 antibody (1:500) in 4 DEG C of reactions overnight, after secondary daily TBS/0.5%Tween 20 washes film 3 times, with HRP labels
37 DEG C of goat anti-rabbit igg secondary antibody incubation 1h), after TBS/0.5%Tween20 washes film 3 times, premix HRP chemiluminescent substrates are added in,
And pass through chemiluminescence gel imaging system and be detected, Fig. 2 B be Western blot detect PTGDS modification excretion body and
Compare the expression result figure of PTGDS, CD63, CD9 and CRTH2 (receptor of PGD2) in excretion body.As shown in Figure 2 B,
The excretion body of PTGDS modifications and label CD9, the CD63 positive expression of control excretion body；Simultaneously it can be found that PTGDS is modified
Excretion body in PTGDS expressing quantities be to present significantly to increase, it was demonstrated that PTGDS be overexpressed adenovirus to interstital stem cell
The modification of excretion body is effective (Fig. 2 B)；
(7) the excretion body of quantitative PCR detection PTGDS modifications can cause increasing for PTGDS gene expressions in tumour cell,
The results are shown in Figure 3 for quantitative PCR detection：PTGDS genes in stomach cancer cell SGC-7901 after the excretion body effect of PTGDS modifications
Expression quantity increasing in conspicuousness, it was demonstrated that interstital stem cell excretion body can carry PTGDS and enter in stomach cancer cell (Fig. 3).
Quantitatively the primer sequence of detection PTGDS is as shown in SEQ ID NO.1~2, specially：
1 primer sequence of table
Embodiment 2
Main material and source difference are as follows：
Cell culture reagent：1640DMEM (Gibco Products), fetal calf serum (Gibco Products), trypsase
(Sigma Products), carbon dioxide incubator (Forma companies), serum free medium (Shanghai Yi Kesai companies)；
Inverted microscope, biomicroscope, superclean bench, desk centrifuge；
24 (6) well culture plates (Corning companies), Transwell migration plates (Corning companies)；
Gastric cancer cell is purchased from Shanghai cell institute of the Chinese Academy of Sciences；
The excretion body of PTGDS modifications has the function of to inhibit external Growth of Gastric migration：
Detailed process is as follows：
(1) influence that the excretion body of PTGDS modifications forms tumour cell body outer clone, the results are shown in Figure 4：By gastric cancer
For cell SGC-7901 cells kind in 6 orifice plates, cell density is 1000/ hole, while adds in 160 μ g/ml's and 320 μ g/ml
It, should after being fixed with paraformaldehyde after the excretion body of PTGDS modifications and the control excretion body of similary concentration carry out stimulation 7 days
Dyed with crystal violet, clone quantity number can be determined that tumour cell growth ability and this inhibiting effect with
PGD2 activities are in dependence.The results show that the excretion body of PTGDS modifications is particularly under 320 μ g/ml concentration conditions, energy
It is enough significantly to inhibit the clonality of SGC-7901 cells, and it is not notable (Fig. 4) to compare excretion body effect；
(2) on tumour cell, external quantity amplification influences the excretion body of PTGDS modifications, and the results are shown in Figure 5：Gastric cancer is thin
Born of the same parents' SGC-7901 cells kind is in 24 orifice plates, and cell density is 5000/ hole, while the PTGDS modifications of 320 μ g/ml of addition is outer
Secreting the control excretion body of body and similary concentration is stimulated.It is acted on for 24 hours in the excretion body of various concentration PTGDS modifications, 48h,
The quantity per hole cell is counted after 72h, the results show that since for 24 hours, compared with negative control and PBS groups, PTGDS modifications
Excretion body inhibits the increase of SGC-7901 cell quantities, and concentration is higher to inhibit more apparent (Fig. 5)；
(3) influence of excretion body external migration to tumour cell of PTGDS modifications, the results are shown in Figure 6, wherein,
Fig. 6 A are：Cell violet staining result after migration；Fig. 6 B are：Counting statistics (the * p of cell after migration<0.05；***p<
0.001)：By SGC-7901 cells kind in 6 plates, cell density is 100000/ hole, and 320 μ g/ are added in after cell is completely adherent
The excretion body of PTGDS modifications and the control excretion body of similary concentration of ml is stimulated.The excretion body stimulation of PTGDS modifications
After 48h, counted after digesting each hole cell by pancreatin, the cell that different groups are stimulated is small in Transwell with 100000 kinds
It (is diluted in room with the 1640DMEM of serum-free), the lower room in Transwell holes adds 500 μ l to contain 10% fetal calf serum
1640DMEM culture solutions are put into incubator and continue to cultivate 12h, take out Transwell plates and are fixed by 4% paraformaldehyde
It is dyed afterwards using crystal violet, will cultivate cell above cell film by cotton swab cleans, and counts the quantity of cell below film, knot
Fruit shows, compared with compareing excretion body, the excretion body of PTGDS modifications is notable must to inhibit SGC-7901 transfer abilities (Fig. 6)；
Embodiment 3
Cell culture reagent：Nude mice (Shanghai Si Laike Company of Animals Ltd.), 1640 DMEM (Gibco Products), tire
Cow's serum (Gibco Products), trypsase (Sigma Products), carbon dioxide incubator (Forma companies), without blood
Clear culture medium (Shanghai Yi Kesai companies), the big wares of 10cm (Corning companies)；
Inverted microscope, biomicroscope, superclean bench, desk centrifuge, HE microsection manufactures and coloring system；
The excretion body of PTGDS modifications inhibits the growth of the subendothelial knurl of Mice Body
(1) by SGC-7901 cells with 105Uniform amount be inoculated in the big wares of 10cm, next day after cell completely it is adherent after
The excretion body and PBS for adding in control excretion body and PTGDS modifications carry out stimulation 48h；
(2) in Shanghai, nude mice 18 is bought by Si Laike Company of Animals Ltd., is randomly assigned 3 groups, and 10 are injected respectively at left side6
Cell quantity be subcutaneously injected control excretion body and PTGDS modification excretion body and the post-stimulatory SGC-7901 cells of PBS, into
The subcutaneous lotus knurl experiment of row；
(3) after subcutaneous tumors are grown 20 days, nude mice is put to death, knurl is taken out and takes pictures, to observe the life of different group knurls
Long status, the results show that excretion body treated the SGC-7901 cells one-tenth knurl ability (as shown in Figure 7) of PTGDS modifications and swelling
The weight of knurl is markedly inferior to other control groups (as shown in Figure 8 A), it was demonstrated that its internal anti-cancer ability (Fig. 7 and Fig. 8 A)；
(4) volume of knurl is continuously measured since being injected cell, as a result as shown in Figure 8 B, is repaiied in 15 days or so PTGDS
The tumor volume of the excretion body processing group of decorations start be less than other control groups, and this trend with knurl growth time not
It is disconnected to increase (Fig. 8 B)；
(5) making of pathological section and the dyeing of HE, the results are shown in Figure 9 after dyeing (arrow are carried out after taking out knurl tissue
The signified vascular site of head), it can be seen that the knurl tissue cellularity of the excretion body processing group of PTGDS modifications is loose and blood
The quantity of pipe is less than other control groups (Fig. 9).
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
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Claims (10)
1. a kind of interstital stem cell excretion body for being overexpressed PTGDS genes, which is characterized in that the overexpression PTGDS genes
The preparation method of interstital stem cell excretion body includes the following steps：
1) PTGDS is overexpressed Adenovirus Transfection interstital stem cell, obtaining PTGDS through screening is overexpressed interstital stem cell；
2) PTGDS that the step 1) obtains is overexpressed interstital stem cell and is seeded to the low sugar DMEM culture mediums containing fetal calf serum
When cell fusion is to 70~80%, the second amplification cultivation is carried out using serum free medium for the first amplification cultivation of middle progress,
Supernatant is collected after 48h, centrifugation removal floating living cells obtains the supernatant of body containing excretion；
3) supernatant of body containing excretion that the step 2) obtains is isolated and purified using sucrose density gradient centrifugation, obtained
Express the interstital stem cell excretion body of PTGDS genes.
2. the interstital stem cell excretion body according to claim 1 for being overexpressed PTGDS genes, which is characterized in that the step
It is rapid 1) in, PTGDS be overexpressed adenovirus with 108Titre transfected.
3. the interstital stem cell excretion body according to claim 1 for being overexpressed PTGDS genes, which is characterized in that step 2)
Containing the fetal calf serum that mass concentration is 10% in the low sugar DMEM culture mediums containing fetal calf serum.
4. the interstital stem cell excretion body according to claim 1 for being overexpressed PTGDS genes, which is characterized in that step 2)
The condition of the centrifugation centrifuges 10min for 300 × g.
5. the interstital stem cell excretion body according to claim 1 for being overexpressed PTGDS genes, which is characterized in that step 3)
Described isolate and purify includes the following steps：
A) supernatant of body containing excretion is centrifuged into 10min in 2000 × g, obtains the supernatant of removal cell fragment；
B) supernatant of the removal cell fragment is centrifuged into 30min in 10000 × g, obtains the supernatant of removal organelle；
C) supernatant of removal organelle that step b) is obtained is subjected to the first concentration, obtains the first concentrate；
D) the first concentrate that step c) is obtained is moved to sucrose/heavy water density pad of 1/4 times of volume, 100000 × g centrifuges 3h,
Sucrose/heavy water layer of bottom is collected, the second concentration is carried out, obtains the second concentrate；
E) the second obtained concentrates of the step d) are washed, obtains being overexpressed between PTGDS genes after filtration sterilization
Matter stem cell excretion body.
6. the interstital stem cell excretion body according to claim 5 for being overexpressed PTGDS genes, which is characterized in that described the
It is the conditional sampling of one concentration and the described second concentration：1000 × g centrifuges 30 min.
7. the interstital stem cell excretion body according to claim 5 or 6 for being overexpressed PTGDS genes, which is characterized in that described
The molecular cut off of first concentration and the described second concentration is 100kDa.
8. the interstital stem cell excretion body according to claim 5 for being overexpressed PTGDS genes, which is characterized in that step d)
The washing is carried out using PBS buffer solution, and the number of the washing is 3 times.
9. a kind of preparation method for the interstital stem cell excretion body for being overexpressed PTGDS genes, includes the following steps：
1) PTGDS is overexpressed Adenovirus Transfection interstital stem cell, obtaining PTGDS through screening is overexpressed interstital stem cell；
2) PTGDS that the step 1) obtains is overexpressed interstital stem cell and is seeded to the low sugar DMEM culture mediums containing fetal calf serum
When cell fusion is to 70~80%, the second amplification cultivation is carried out using serum free medium for the first amplification cultivation of middle progress,
Supernatant is collected after 48h, centrifugation removal floating living cells obtains the supernatant of the body containing excretion；
3) supernatant of the body containing excretion that the step 2) obtains is isolated and purified using sucrose density gradient centrifugation, obtained
It is overexpressed the interstital stem cell excretion body of PTGDS genes.
10. the interstital stem cell excretion body of PTGDS genes or claim 9 institute are overexpressed described in claim 1~8 any one
The interstital stem cell excretion body of overexpression PTGDS genes that preparation method obtains is stated in prevention or treatment gastric cancer medicament is prepared
Using.
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| CN113088496A (en) * | 2021-03-19 | 2021-07-09 | 广州远想生物科技有限公司 | EGF mesenchymal stem cell exosome and preparation method and application thereof |
| CN115025234A (en) * | 2021-06-22 | 2022-09-09 | 姜海涛 | Stomach targeting drug-loaded exosome, application thereof and drug for treating stomach diseases |
| CN114540294A (en) * | 2022-03-14 | 2022-05-27 | 上海交通大学 | Preparation and application of stem cell exosome for delivering gene drugs to tumor site |
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