CN108251378B - A kind of interstital stem cell excretion body and its preparation method and application being overexpressed PTGDS gene - Google Patents
A kind of interstital stem cell excretion body and its preparation method and application being overexpressed PTGDS gene Download PDFInfo
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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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- A—HUMAN NECESSITIES
- 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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- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The present invention relates to a kind of interstital stem cell excretion bodies and its preparation method and application for being overexpressed PTGDS gene, belong to gastric cancer medicament preparation technical field.It is provided by the invention be overexpressed PTGDS gene interstital stem cell excretion body, be using PTGDS be overexpressed Adenovirus Transfection interstital stem cell after, cultivated, isolate and purify after obtain.The interstital stem cell excretion body efficient target stomach cancer cell provided by the invention for being overexpressed PTGDS gene 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 preparation technical fields, and in particular to a kind of interstitial for being overexpressed PTGDS gene is dry thin
It is extracellular to secrete body and its preparation method and application.
Background technique
Gastric cancer is the fifth-largest common malignant tumour in the world, and the death rate occupies third position, and in China, gastric cancer is high
The third position for occupying most common cancer has been the second largest Cancer death reason.Clinical treatment gastric cancer medicament mainly has 5- fluorine at present
The chemotherapeutics such as uracil, these drugs can inhibit the progress of tumour significantly, but since it is to the special of tumor tissues
Property is poor, and has very strong side effect.Therefore, we urgently need a kind of not only grown with anti-gastric cancer cell 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 biology below
Process: the phospholipase A on cell membrane is converted to arachidonic acid, catalysis of the latter in Cycloxygenase (including COX-1 and COX-2)
Under, be broken down into unstable intermediate product prostaglandin H2, prostaglandin H2 is again by three kinds of different enzymes: prostaglandin D is closed
At 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 is overexpressed PTGDS in tumour cell can to efficiently control external gastric cancer thin
The growth of born of the same parents, (number of patent application: 201611203449.3), but the prior art still lacks practical energy to the potentiality with treatment gastric cancer
The drug of enough efficient targeted therapy gastric cancers.
Summary of the invention
The purpose of the present invention is to provide a kind of interstital stem cell excretion bodies and preparation method thereof for being overexpressed PTGDS gene
And application.The interstital stem cell excretion body efficient target stomach cancer cell provided by the invention for being overexpressed PTGDS gene, 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 gene, the overexpression PTGDS genes
Interstital stem cell excretion body preparation method the following steps are included:
1) PTGDS is overexpressed Adenovirus Transfection interstital stem cell, obtains PTGDS through screening and is overexpressed interstital stem cell；
2) PTGDS that the step 1) obtains is overexpressed interstital stem cell and is seeded to the training of the low sugar DMEM 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 for the body containing excretion that the step 2) obtains is isolated and purified using sucrose density gradient centrifugation,
Obtain the interstital stem cell excretion body for being overexpressed PTGDS gene.
In the present invention, in the step 1), PTGDS is overexpressed adenovirus with 108Titre transfected.
In the present invention, the tire for being 10% containing mass concentration in the low sugar DMEM culture medium containing fetal calf serum described in step 2)
Cow's serum.
Preferably, the condition of the step 2) centrifugation is that 300 × g is centrifuged 10min.
Preferably, step 3) it is described isolate and purify the following steps are included:
A) supernatant of body containing excretion is centrifuged 10min in 2000 × g, obtains the supernatant of removal cell fragment；
B) supernatant of the removal cell fragment is centrifuged 30min in 10000 × g, obtains the supernatant of removal organelle；
C) supernatant for the removal organelle that step b) is obtained is subjected to the first concentration, obtains the first concentrate；
D) the first concentrate for obtaining step c) moves to the 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 concentrate of the step d) is washed, obtains being overexpressed PTGDS gene after filtration sterilization
Interstital stem cell excretion body.
The conditional sampling that preferably, first concentration is concentrated with described second are as follows: 1000 × g is centrifuged 30min.
Preferably, the molecular cut off of first concentration and second concentration is 100kDa.
Preferably, the step d) washing is carried out using PBS buffer solution, and the number of the washing is 3 times.
The present invention also provides a kind of preparation methods of interstital stem cell excretion body for being overexpressed PTGDS gene, including with
Lower step:
1) PTGDS is overexpressed Adenovirus Transfection interstital stem cell, obtains PTGDS through screening and is overexpressed interstital stem cell；
2) PTGDS that the step 1) obtains is overexpressed interstital stem cell and is seeded to the training of the low sugar DMEM 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, is obtained
To the interstital stem cell excretion body for being overexpressed PTGDS gene
The present invention also provides the interstital stem cell excretion body of PTGDS gene or above-mentioned is overexpressed described in above-mentioned technical proposal
The interstital stem cell excretion body for the overexpression PTGDS gene that preparation method described in technical solution obtains is in preparation prevention or treatment stomach
Application in cancer drug.
The present invention provides a kind of interstital stem cell excretion bodies for being overexpressed PTGDS gene.Overexpression provided by the invention
The interstital stem cell excretion body of PTGDS gene 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 gene can efficient target stomach cancer cell, inhibit gastric cancer
The growth and migration of cell, and then inhibit tumour growth.Test result shows overexpression PTGDS gene 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.
Detailed description of the invention
Fig. 1 is the interstital stem cell form and GFP (virus transfection effect after the Adenovirus Transfection that the embodiment of the present invention 1 provides
The labelled protein of rate) fluorescence photo (400 times)；
Fig. 2 is the interstital stem cell excretion body and the unloaded gland of control for the overexpression PTGDS gene that the embodiment of the present invention 1 provides
The separation qualification result figure of the excretion body of virus transfection；
Fig. 3 is the quantitative PCR inspection of the interstital stem cell excretion body for the overexpression PTGDS gene that the embodiment of the present invention 1 provides
Survey result figure；
Fig. 4 is that the interstital stem cell excretion body for the overexpression PTGDS gene 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 for the overexpression PTGDS gene 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 for the overexpression PTGDS gene 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 for the overexpression PTGDS gene 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 tumor weight and growth curve that the embodiment of the present invention 3 provides；
Fig. 9 is the interstital stem cell excretion body for the overexpression PTGDS gene that the embodiment of the present invention 3 provides 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 gene, the overexpression PTGDS genes
Interstital stem cell excretion body preparation method the following steps are included:
1) PTGDS is overexpressed Adenovirus Transfection interstital stem cell, obtains PTGDS through screening and is overexpressed interstital stem cell；
2) PTGDS that the step 1) obtains is overexpressed interstital stem cell and is seeded to the training of the low sugar DMEM 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, is obtained
To the interstital stem cell excretion body for being overexpressed PTGDS gene.
The present invention provides a kind of interstital stem cell excretion body for being overexpressed PTGDS gene, the excretions of abbreviation PTGDS modification
Body, the PTGDS gene are prostaglandin D2 synzyme.
PTGDS is overexpressed Adenovirus Transfection interstital stem cell by the present invention, and it is dry thin to obtain PTGDS overexpression interstitial through screening
Born of the same parents.The present invention, which 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 mistake of hundred Bioisystech Co., Ltd, Australia is such as answered purchased from Suzhou
Express adenovirus.The present invention is normal using those skilled in the art to the source of the interstital stem cell also not special restriction
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) 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 special restriction to the method for the transfection, 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 PTGDS be overexpressed interstital stem cell screening technique it is preferred specifically: with GFP be 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 reaction PTGDS.
After obtaining PTGDS overexpression interstital stem cell, the present invention preferably passes on PTGDS overexpression interstital stem cell
Culture is saved after more preferably passing on 3~5 times, spare.The present invention does not have special limit to the condition of the secondary culture
It is fixed, using trypsin digestion cell secondary culture method well known to those skilled in the art.
After obtaining PTGDS overexpression interstital stem cell, PTGDS overexpression interstital stem cell is seeded to ox containing tire by the present invention
The first amplification cultivation is carried out in the low sugar DMEM culture medium 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, the fetal calf serum for being 10% containing mass concentration in the low sugar DMEM culture medium containing fetal calf serum.The present invention is to described low
The composition of sugared DMEM culture medium is not particularly limited, using conventional commercial low sugar DMEM culture medium well known to those skilled in the art
?.The present invention preferably when cell fusion is to 75%, carries out the second amplification cultivation using serum free medium.In the present invention
In, the condition of the centrifugation removal floating living cells is preferably 300 × g centrifugation 10min.
After obtaining the supernatant of body containing excretion, the present invention separates the supernatant of body containing excretion using sucrose density gradient centrifugation
Purifying obtains the interstital stem cell excretion body for being overexpressed PTGDS gene.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 10min in 2000 × g, obtains the supernatant of removal cell fragment；
B) supernatant of the removal cell fragment is centrifuged 30min in 10000 × g, obtains the supernatant of removal organelle；
C) supernatant for the removal organelle that step b) is obtained is subjected to the first concentration, obtains the first concentrate；
D) the first concentrate for obtaining step c) moves to the 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 concentrate of the step d) is washed, obtains being overexpressed PTGDS gene after filtration sterilization
Interstital stem cell excretion body.
The present invention will contain excretion body supernatant and be centrifuged 10min in 2000 × g, obtain the supernatant of removal cell fragment.In this hair
In bright, the centrifugation is preferably carried out under the conditions of 4 DEG C.
After obtaining the supernatant of removal cell fragment, the supernatant for removing cell fragment is centrifuged by the present invention 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 the sucrose/heavy water density of 1/4 times of volume by the present invention
Pad, 100000 × g are centrifuged 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 are preferably centrifuged 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, be preferably diluted to without apparent precipitating,
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 gene.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 preferably uses sterilised membrane filter to carry out, 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 gene preferably carries out under the conditions of -70 DEG C
It saves.It obtains after being overexpressed the interstital stem cell excretion body of PTGDS gene, the present invention is between the overexpression PTGDS gene
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 kit.
The present invention also provides a kind of preparation methods of interstital stem cell excretion body for being overexpressed PTGDS gene, including with
Lower step:
1) PTGDS is overexpressed Adenovirus Transfection interstital stem cell, obtains PTGDS through screening and is overexpressed interstital stem cell；
2) PTGDS that the step 1) obtains is overexpressed interstital stem cell and is seeded to the training of the low sugar DMEM 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, is obtained
To the interstital stem cell excretion body for being overexpressed PTGDS gene.
The detailed preparation parameter that the present invention is overexpressed the preparation method of the interstital stem cell excretion body of PTGDS gene 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 gene or above-mentioned is overexpressed described in above-mentioned technical proposal
The interstital stem cell excretion body for the overexpression PTGDS gene that preparation method described in technical solution obtains is in preparation prevention or treatment stomach
Application in cancer drug.In the present invention, the dosage form of the drug is preferably liquid preparation.
Combined with specific embodiments below to a kind of interstital stem cell excretion body for being overexpressed PTGDS gene of the present invention
And its preparation method and application be further described in detail, technical solution of the present invention includes but is 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 company), serum free medium (Shanghai Yi Kesai company；
Inverted microscope, fluorescence microscope, biomicroscope, electron microscope, superclean bench, desk centrifuge；
PTGDS is overexpressed adenovirus vector (answering hundred Bioisystech Co., Ltd, Australia in Suzhou), heavy water (D2O, upper SeaBird match are public
Department), it analyzes pure sucrose (Guangzhou Chemical Reagent Factory), rabbit-anti people CD9 antibody (Bioworld Technology company, the U.S.), rabbit
Anti-human CD63 antibody (Epitomics company, the U.S.), the mountain of BCA protein quantification kit, horseradish peroxidase (HRP) label
Goat anti-rabbit igg secondary antibody (Beijing health is ShiJi Co., Ltd), HRP chemiluminescent substrate, 100-kDaMWCO ultra-filtration centrifuge tube, 0.22 μm
Sterilised membrane filter (Millipore company, the U.S.)；Transmission electron microscope (FEITecnai12, Philips company)；Quantitative PCR examination
Agent (Beijing Cwbio company).
It is overexpressed the acquisition of the interstital stem cell excretion body (the excretion body of PTGDS modification) of PTGDS gene
(1) by interstital stem cell in 37 DEG C, 5%CO2Culture, cultivates successful interstital stem cell in saturated humidity incubator
Form is shown in Fig. 1.
(2) PTGDS is overexpressed adenovirus and its control 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).In conjunction with white light photo, transfection efficiency is in 80% or more (figure
1)。
(3) in selection growth conditions good 3~5 generation PTGDS, is overexpressed interstital stem cell and first uses containing 10% fetal calf serum
Low sugar DMEM culture medium culture is changed serum free medium culture when cell fusion is to 70%~80%, is collected in culture after 48h
Clearly, 300 × g is centrifuged 10min to remove the living cells of floating, for be overexpressed PTGDS gene interstital stem cell excretion body (with
It is lower with PTGDS modification excretion body surface show) separation.
(4) the interstital stem cell supernatant of collection is centrifuged 10min in 4 DEG C, 2000 × g, to remove cell fragment；It collects
It is centrifuged 30min in 4 DEG C, 10000 × g after supernatant, to remove organelle；Supernatant is transferred to 100-kDaMWCO ultra-filtration centrifuge tube,
4 DEG C, 1000 × g centrifugation 30min be concentrated；Concentrate is slowly moved to 5ml30% sucrose/heavy water density pad (ρ=
1.210g/cm3), 4 DEG C, 100000 × g centrifugation 3h；Bottom 5ml sucrose/heavy water layer (containing exosome) is collected, is added after PBS dilution
Enter in 100-kDaMWCO ultra-filtration centrifuge tube, 4 DEG C, 1000 × g centrifugation 30min, PBS washing 3 times；Finally with 0.22 μm of sterile filter
Film filtration sterilization saves in -70 DEG C after packing, 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 modification 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 mixing well, in room
After temperature stands 5min, copper mesh edge residual liquid is gently sucked with filter paper, and then copper mesh is buckled in 30g/L phosphotungstic acid
(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
According to the excretion body of PTGDS modification and the separation qualification result for compareing excretion body are as shown in Figure 2, wherein Fig. 2A are as follows: transmission electron microscope
Observe its form (scale 100nm) result figure.As shown in Figure 2 A, the excretion body by PTGDS modification and control excretion body are straight
Diameter is about 100nm capsule balloon-shaped structure；
(6) Westernblot detects the excretion body of PTGDS modification and the surface markers albumen of control excretion body: preparing
15%SDS-PAGE running gel is added 1/4 after sufficiently cracking the PTGDS of the said extracted excretion body modified and control excretion body
5 × SDS sample-loading buffer of volume, boils 5min, and by 200 μ g protein total amount loadings, electrotransfer (350mA, 120min) will
Protein is gone on pvdf membrane, closes 1h with the TBS/T room temperature of the skim milk containing 50g/L, respectively with rabbit-anti people CD9 antibody and rabbit
Anti-human CD81 antibody (1:500) overnight, after secondary daily TBS/0.5%Tween 20 washes film 3 times, marks in 4 DEG C of reactions with HRP
37 DEG C of incubation 1h of goat anti-rabbit igg secondary antibody), after TBS/0.5%Tween20 washes film 3 times, premix HRP chemiluminescent substrate is added,
And detected by chemiluminescence gel imaging system, 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 modification 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 quantity be present increase significantly, 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 modification can cause increasing for PTGDS gene expression in tumour cell,
Quantitative PCR detection result is as shown in Figure 3: PTGDS gene in the stomach cancer cell SGC-7901 after the excretion body effect of PTGDS modification
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).
The primer sequence of quantitative detection PTGDS as shown in NO.1~2 SEQ ID, specifically:
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 company), serum free medium (Shanghai Yi Kesai company)；
Inverted microscope, biomicroscope, superclean bench, desk centrifuge；
24 (6) well culture plates (Corning company), Transwell migration plate (Corning company)；
Gastric cancer cell is purchased from Chinese Academy of Sciences Shanghai cell institute；
The excretion body of PTGDS modification has the function of inhibiting external Growth of Gastric migration:
Detailed process is as follows:
(1) influence that the excretion body of PTGDS modification forms tumour cell body outer clone, as a result as shown in Figure 4: by gastric cancer
For cell SGC-7901 cell kind in 6 orifice plates, cell density is 1000/ hole, while being added 160 μ g/ml's and 320 μ g/ml
After the excretion body of PTGDS modification and the control excretion body of same concentration carry out stimulation 7 days, answered after being fixed with paraformaldehyde
Dyed with crystal violet, clone quantity number can be determined that tumour cell growth ability and this inhibiting effect with
PGD2 activity is in dependence.The results show that the excretion body of PTGDS modification is especially under 320 μ g/ml concentration conditions, energy
Enough clonalities for inhibiting SGC-7901 cell significantly, and it is not significant (Fig. 4) to compare excretion body effect；
(2) the excretion body of PTGDS modification expands the external quantity of tumour cell and influences, as a result as shown in Figure 5: gastric cancer is thin
Born of the same parents' SGC-7901 cell kind is in 24 orifice plates, and cell density is 5000/ hole, while the PTGDS modification of 320 μ g/ml of addition is outer
The control excretion body for secreting body and same concentration is stimulated.It is acted on for 24 hours in the excretion body of various concentration PTGDS modification, 48h,
The quantity of every hole cell is counted after 72h, the results show that since for 24 hours, compared with negative control and PBS group, PTGDS modification
Excretion body inhibits the increase of SGC-7901 cell quantity, and the higher inhibition of concentration is more obvious (Fig. 5)；
(3) influence of the excretion body of PTGDS modification to the external migration of tumour cell, as a result as shown in Figure 6, wherein
Fig. 6 A are as follows: cell violet staining result after migration；Fig. 6 B are as follows: counting statistics (p < 0.05 * of cell after migration；***p<
0.001): by SGC-7901 cell kind in 6 plates, cell density is 100000/ hole, and 320 μ g/ are added after cell is completely adherent
The excretion body of PTGDS modification and the control excretion body of same concentration of ml is stimulated.The excretion body stimulation of PTGDS modification
It after 48h, is 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 with the 1640DMEM of serum-free) in room, the lower room in the hole Transwell adds 500 μ l to contain 10% fetal calf serum
1640DMEM culture solution is put into incubator and continues to cultivate 12h, takes out Transwell plate and is fixed by 4% paraformaldehyde
It is dyed afterwards using crystal violet, cell above cell film will be cultivated by cotton swab and cleaned, the quantity of cell below film, knot are counted
Fruit shows, compared with compareing excretion body, the excretion body of PTGDS modification is significant must to inhibit SGC-7901 transfer ability (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 company), without blood
Clear culture medium (Shanghai Yi Kesai company), the big ware of 10cm (Corning company)；
Inverted microscope, biomicroscope, superclean bench, desk centrifuge, HE microsection manufacture and coloring system；
The excretion body of PTGDS modification inhibits the growth of the subendothelial tumor of Mice Body
(1) by SGC-7901 cell with 105Uniform amount be inoculated in the big ware of 10cm, next day after cell completely it is adherent after
The excretion body and PBS that control excretion body and PTGDS modification is added carry out stimulation 48h；
(2) Yu Shanghai Si Laike Company of Animals Ltd. buys nude mice 18, is randomly assigned 3 groups, injects 10 respectively at left side6
Cell quantity subcutaneous injection control excretion body and PTGDS modification excretion body and the post-stimulatory SGC-7901 cell 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 cell one-tenth knurl ability (as shown in Figure 7) of PTGDS modification and swelling
The weight (as shown in Figure 8 A) of tumor is lower than other control groups significantly, it was demonstrated that its intracorporal anti-cancer ability (Fig. 7 and Fig. 8 A)；
(4) continuously as a result as shown in Figure 8 B the volume of measurement knurl was repaired in 15 days or so PTGDS since injecting cell
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) production of pathological section and the dyeing of HE are carried out after taking out knurl tissue, result (arrow as shown in Figure 9 after dyeing
The signified vascular site of head), it can be seen that the knurl tissue cellularity of the excretion body processing group of PTGDS modification is loose and blood
The quantity of pipe is less than other control groups (Fig. 9).
The above is only a 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 are also answered
It is considered as protection scope of the present invention.
Sequence table
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<213>artificial sequence (Artificial Sequence)
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Claims (5)
1. a kind of application of interstital stem cell excretion body for being overexpressed PTGDS gene in preparation prevention or treatment gastric cancer medicament,
It is characterized in that, it is described be overexpressed PTGDS gene interstital stem cell excretion body preparation method the following steps are included:
1) PTGDS is overexpressed Adenovirus Transfection interstital stem cell, obtains PTGDS through screening and is overexpressed interstital stem cell；PTGDS
Adenovirus is overexpressed with 108Titre transfected；
2) PTGDS that the step 1) obtains is overexpressed interstital stem cell and is seeded to the low sugar DMEM culture medium containing fetal calf serum
The first amplification cultivation of middle progress carries out the second amplification cultivation using serum free medium when cell fusion is to 70~80%,
Supernatant is collected after 48h, centrifugation removal floating living cells obtains the supernatant of body containing excretion；The condition of the centrifugation is 300 × g centrifugation
10min；
3) supernatant of body containing excretion that the step 2) obtains is isolated and purified using sucrose density gradient centrifugation, is obtained
Express the interstital stem cell excretion body of PTGDS gene；
It is described isolate and purify the following steps are included:
A) supernatant of body containing excretion is centrifuged 10min in 2000 × g, obtains the supernatant of removal cell fragment；
B) supernatant of the removal cell fragment is centrifuged 30min in 10000 × g, obtains the supernatant of removal organelle；
C) supernatant for the removal organelle that step b) is obtained is subjected to the first concentration, obtains the first concentrate；
D) the first concentrate for obtaining step c) moves to the sucrose/heavy water density pad of 1/4 times of volume, and 100000 × g is centrifuged 3h,
Sucrose/heavy water layer of bottom is collected, the second concentration is carried out, obtains the second concentrate；
E) the second obtained concentrate of the step d) is washed, obtains being overexpressed between PTGDS gene after filtration sterilization
Matter stem cell excretion body.
2. application according to claim 1, which is characterized in that the low sugar DMEM culture medium containing fetal calf serum described in step 2)
In containing mass concentration be 10% fetal calf serum.
3. application according to claim 1, which is characterized in that the conditional sampling of first concentration and second concentration
Ground are as follows: 1000 × g is centrifuged 30min.
4. application according to claim 1, which is characterized in that the retention molecule of first concentration and second concentration
Amount is 100kDa.
5. application according to claim 1, which is characterized in that the step d) washing is carried out using PBS buffer solution, described
The number of washing is 3 times.
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