CN108795860A - Lyopgized Nocardia rubra-cell Wall Skeleton is as CD8+The purposes of T cell enhancer of proliferation - Google Patents
Lyopgized Nocardia rubra-cell Wall Skeleton is as CD8+The purposes of T cell enhancer of proliferation Download PDFInfo
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Abstract
The present invention relates to Lyopgized Nocardia rubra-cell Wall Skeletons as CD8+T cell enhancer of proliferation, CD8+T cell function of surface molecule CD25 and CD69 expression accelerating agent, CD8+T cell intracellular granzyme B and perforin expression accelerating agent or CD8+T cell TNF-α expresses the purposes of accelerating agent.
Description
Technical field
The invention belongs to field of medicaments, and in particular to Lyopgized Nocardia rubra-cell Wall Skeleton is as CD8+T cell proliferation promotees
Into the purposes of agent.
Background technology
Nocard's bacillus is polymorphic, there is that Nocardia is spherical, rod-shaped, Filamentous, thalline size 0.6 × (3~4) micron,
Without motion, some strains are in weak acid-resisting, and obligate aerobic, nutritional requirement is general.Have after being cultivated 3 days on plain agar tablet
Visible colonies, bacterium colony projection after 7~10 days, after aerial hyphae is formed, surface is in villiform.Bacterium colony not of the same race has yellow, orange, red
Or the secondary colour of these pigments.G+C gram molecule contents in DNA are 60~72%.It is mostly saprophytic bacteria, is present in soil.
Nocardia rubra (Nocardiarubra) is one such actinomyces.Nocardia rubra thalline is fermented, cell is broken
Lyopgized Nocardia rubra-cell Wall Skeleton (hereinafter referred to as Nr-CWS or N-CWS) can be made after broken, proteasome degradation.
Invention content
Present invention discover that Lyopgized Nocardia rubra-cell Wall Skeleton can promote CD8+T cell proliferation promotes CD8+T cell table
The expression of face functional molecular CD25 and CD69 promote CD8+The expression of T cell intracellular granzyme B and perforin and promotion CD8+
The expression of T cell TNF-α, and then complete the present invention.
According to an aspect of the present invention, the present invention provides Lyopgized Nocardia rubra-cell Wall Skeleton and is preparing for promoting
CD8+Purposes in the drug of T cell proliferation.According to the present invention, the drug can be by promoting CD8+T cell anti-proliferative treatment with
CD8+The relevant disease of T cell, including but not limited to:Such as body local organization caused by the cause pathogeny imcrobe infection of virus etc.
Infection or lesion and autoimmune disease.
According to another aspect of the present invention, the present invention provides Lyopgized Nocardia rubra-cell Wall Skeleton as non-treatment purpose
CD8+The purposes of T cell enhancer of proliferation.According to the present invention, " non-treatment purpose " for example promotes CD8 in vitro+T cell
Proliferation.
According to another aspect of the present invention, the present invention provides a kind of external promotion CD8+The method of T cell proliferation,
In, give the CD8 of in vitro culture+T cell Lyopgized Nocardia rubra-cell Wall Skeleton.It is preferred that Lyopgized Nocardia rubra-cell Wall Skeleton
A concentration of 1-60 μ g/ml, further preferred 2-30 μ g/ml, more preferable 3-15 μ g/ml.
According to another aspect of the present invention, the present invention provides Lyopgized Nocardia rubra-cell Wall Skeleton and is preparing for promoting
CD8+T cell function of surface molecule CD25, CD8+T cell function of surface molecule CD69, CD8+T cell intracellular granzyme B or CD8+
Purposes in the drug of T cell intracellular perforin expression.According to the present invention, the drug can be by promoting CD8+T cell table
Face functional molecular CD25, CD8+T cell function of surface molecule CD69, CD8+T cell intracellular granzyme B or CD8+T cell intracellular is worn
Hole element expression treatment and CD8+The relevant disease of T cell, including but not limited to:Such as the cause pathogeny imcrobe infection of virus etc. causes
Body local tissue infection or lesion and autoimmune disease.
According to another aspect of the present invention, the present invention provides Lyopgized Nocardia rubra-cell Wall Skeleton as non-treatment purpose
CD8+T cell function of surface molecule CD25, CD8+T cell function of surface molecule CD69, CD8+T cell intracellular granzyme B or
CD8+The purposes of T cell intracellular perforin expression accelerating agent.According to the present invention, " non-treatment purpose " for example promotes in vitro
CD8+T cell function of surface molecule CD25, CD8+T cell function of surface molecule CD69, CD8+T cell intracellular granzyme B or CD8+
T cell intracellular perforin expression.
According to another aspect of the present invention, the present invention provides a kind of external promotion CD8+T cell function of surface molecule
CD25、CD8+T cell function of surface molecule CD69, CD8+T cell intracellular granzyme B or CD8+T cell intracellular perforin expression
Method, wherein give the CD8 of in vitro culture+T cell Lyopgized Nocardia rubra-cell Wall Skeleton.It is preferred that nocardia rubra cell
A concentration of 1-60 μ g/ml of wall skeleton, further preferred 2-30 μ g/ml, more preferable 3-15 μ g/ml.
According to another aspect of the present invention, the present invention provides Lyopgized Nocardia rubra-cell Wall Skeleton and is preparing for promoting
CD8+Purposes in the drug of T cell TNF-α expression.According to the present invention, the drug can be by promoting CD8+T cell TNF-α
Expression treatment and CD8+The relevant disease of T cell, including but not limited to:Such as machine caused by the cause pathogeny imcrobe infection of virus etc.
Body local tissue infection or lesion and autoimmune disease.
According to another aspect of the present invention, the present invention provides Lyopgized Nocardia rubra-cell Wall Skeleton as non-treatment purpose
CD8+T cell TNF-α expresses the purposes of accelerating agent.According to the present invention, " non-treatment purpose " for example promotes CD8 in vitro+
T cell TNF-α is expressed.
According to another aspect of the present invention, the present invention provides a kind of external promotion CD8+The side of T cell TNF-α expression
Method, wherein give the CD8 of in vitro culture+T cell Lyopgized Nocardia rubra-cell Wall Skeleton.It is preferred that nocardia rubracell wall
A concentration of 1-60 μ g/ml of skeleton, further preferred 2-30 μ g/ml, more preferable 3-15 μ g/ml.
It will be understood by those skilled in the art that Lyopgized Nocardia rubra-cell Wall Skeleton can it is commercially available or according to this field
The various methods known are prepared.It is preferred that nocardia rubra is that (it is micro- that it was preserved in China on 2 5th, 2002 to Nr-8206
Biological inoculum preservation administration committee common micro-organisms center, deposit number are CGMCC NO.0712).For example, can pass through by
Removal DNA, DNA, RNA, protein, lipoid, are prepared Lyopgized Nocardia rubra-cell Wall Skeleton after nocardia rubra fermentation,
Its principle active component is muramic acid, polysaccharide and mucopeptide, and molecular weight is about 2-20KD.
The present invention confirms that Lyopgized Nocardia rubra-cell Wall Skeleton can promote CD8 for the first time by experiment+T cell proliferation,
Promote CD8+T cell function of surface molecule CD25, CD8+T cell function of surface molecule CD69, CD8+T cell intracellular granzyme B or
CD8+The expression of T cell intracellular perforin and promotion CD8+The expression of T cell TNF-α, so that nocardia rubra is thin
Cell wall skeleton can be used to treat in vivo and CD8+The related disease of T cell, and can be used to promote CD8 in vitro+T is thin
Born of the same parents' proliferation promotes CD8+T cell function of surface molecule CD25, CD8+T cell function of surface molecule CD69, CD8+T cell intracellular
Granzyme B or CD8+The expression of T cell intracellular perforin and promotion CD8+The expression of T cell TNF-α.
Description of the drawings
Fig. 1 Lyopgized Nocardia rubra-cell Wall Skeletons promote CD8+T cell is proliferated:Figure 1A is 24 hours, and Figure 1B is 48 hours
Fig. 2 Lyopgized Nocardia rubra-cell Wall Skeletons promote CD8+The expression of T cell function of surface molecule CD25 and CD69:
Fig. 2A 1 is 24 hours streaming figures;Fig. 2A 2 is 24 hours CD25 and CD69 expression comparisons;Fig. 2 B1 are 48 hours streaming figures;Fig. 2 B2
For 48 hours CD25 and CD69 expression comparisons;Fig. 2 C are 24 hours TRAIL and FasL expression comparisons;Fig. 2 D are 48 hours TRAIL
It expresses and compares with FasL
Fig. 3 Lyopgized Nocardia rubra-cell Wall Skeletons promote CD8+The expression of T cell intracellular granzyme B and perforin:Figure
3A1 is 24 hours streaming figures;Fig. 3 A2 are that 24 hours granzyme Bs and perforin expression compare;Fig. 3 B1 are 48 hours streaming figures;Figure
3B2 is that 48 hours granzyme Bs and perforin expression compare
Fig. 4 Lyopgized Nocardia rubra-cell Wall Skeletons promote CD8+The expression of T cell TNF-α
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.Furthermore, it is to be understood that after having read recorded content of the invention, this field skill
Art personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within limited range of the present invention.
Embodiment 1.Nr-CWS promotes CD8+T cell is proliferated
1. mouse CD8+T cell is isolated and purified, is cultivated
(1) sterile separating mouse (cleaning grade C57BL/6 mouse, 6-8 week old, female, 18-20 grams of weight, be purchased from Shanghai this
Lake experimental animal Co., Ltd) spleen, prepare single cell suspension;Cell is discarded supernatant to obtain after 1000rpm/min centrifugations 10min
Precipitation;Gained spleen cell precipitation is suspended with 2ml erythrocyte cracked liquids, on ice splitting erythrocyte 5min, and it is slow that the cold PBS of 10ml are added
Fliud flushing, 1000rpm/min discard supernatant to obtain cell precipitation after centrifuging 5min;It is resuspended in 2ml magnetic bead sorting buffer solutions, adjustment is thin
Born of the same parents a concentration of 1 × 108/ml。
(2) according to StemcellEasySepTM mouse CD8+T cell isolation Kit Beads enrichments are used and are said
Bright book operating procedure, sterile sorting CD8+T cell and be about 92.93 ± 1.63% with flow cytometer detection separating purity.
(3) with addition 10% heat-inactivated fetal bovine serum (FBS) 1640 culture mediums of complete RPMI (2mM L-Glutamines,
50 μM of beta -mercaptoethanols, 100U/ml penicillin, 100 μ g/ml streptomysins, 100 μ g/ml kanamycins) suspension fresh separated
CD8+T cell, by 1 × 106/ ml cell densities are inoculated with 96 orifice plates (coating anti-CD3 antibody in advance), per 100 μ l of hole, and
Anti-CD28 antibody is added in culture medium;In 37 DEG C, 5%CO2Incubator in cultivate.
2.Nr-CWS promotes CD8+T cell is proliferated
CD8+T cell presses 1 × 106/ ml cell densities are inoculated with 96 orifice plates, per 100 μ l of hole.In 37 DEG C, 5%CO2Incubator
After middle culture 2 hours, experimental group addition is respectively 1.875 μ g/ml, 3.75 μ g/ml, 7.5 μ g/ with the concentration that culture solution dissolves
The Nr-CWS (Liaoning Ge Ruishite Biology Pharmacy Co., Ltd, similarly hereinafter) of ml, 15 μ g/ml, 30 μ g/ml, 60 μ g/ml, per hole 100
μ l, each concentration set 3 multiple holes.RPMI 1640 culture mediums are added in control group, per 100 μ l of hole, if 3 multiple holes.37 DEG C, 5%
CO2Incubator in cultivate 24 hours and 48 hours.At last 4 hours of each detection time point, 20 μ l MTS/ are added per hole
PMS mixed liquors (96Aqueous One Solution Cell Proliferation Assay kit).It terminates
After culture, the absorbance in each hole, reference wavelength 600nm are measured at enzyme-linked immunosorbent assay instrument OD490nm.
As a result (see Fig. 1, wherein Figure 1A is 24 hours, and Figure 1B is 48 hours) display, Nr-CWS a concentration of 7.5 μ g/ml,
When 15 μ g/ml, 30 μ g/ml, 60 μ g/ml, CD8 is handled+After T cell 24 hours, CD8+T cell proliferation is notable compared with the control group
Increase (* p<0.05 or * * p<0.01);Nr-CWS a concentration of 1.875 μ g/ml, 3.75 μ g/ml, 7.5 μ g/ml, 15 μ g/ml,
When 30 μ g/ml, 60 μ g/ml, CD8 is handled+After T cell 48 hours, CD8+T cell proliferation compared with the control group significantly increase (#p<
0.001);Compared with when a concentration of 7.5 μ g/ml, Nr-CWS handles CD8 in a concentration of 15 μ g/ml+After T cell 48 hours,
CD8+The significantly raising of T cell proliferation (△p<0.001)。
Embodiment 2.Nr-CWS promotes CD8+The expression of T cell function of surface molecule CD25, CD69
CD8+T cell presses 1 × 106/ ml cell densities are inoculated with 96 orifice plates, per 100 μ l of hole, in 37 DEG C, 5%CO2Incubator
After middle culture 2 hours, the Nr-CWS for a concentration of 15 μ g/ml that experimental group addition culture solution dissolves, per 100 μ l of hole, if 3 are multiple
Hole.RPMI 1640 culture mediums are added in control group, per 100 μ l of hole, if 3 multiple holes.The control group that is unstained makees same place with control group
Reason, but do not dye.In 37 DEG C, 5%CO2Incubator in cultivate 24 hours and 48 hours.Collect each group CD8+T cell is marked respectively
Remember anti-CD8a, anti-CD25, anti-CD69, anti-TRAIL and anti-FasL streaming fluorescence antibody, then washes away not
Combination dye prepares the cell suspension of 500 μ l.Using flow cytometer detection CD8+T cell function of surface molecule CD25, CD69, TRAIL
With the expression of FasL.Wherein, at least 10000 cells are collected using BD FACSDiva softwares.
As a result it is shown (see Fig. 2), Nr-CWS handles CD8+T cell (Fig. 2A 1 and Fig. 2A 2), CD8 after 24 hours+T cell table
Face functional molecular CD25 and CD69 expression significantly increases (* p compared with the control group<0.05);Processing 48 hours after (Fig. 2 B1 and
Fig. 2 B2), CD8+T cell function of surface molecule CD25 and CD69 expression significantly increases (* p compared with the control group<0.05);Nothing
By be processing 24 hours after (Fig. 2 C) or (Fig. 2 D) after 48 hours, CD8+T cell function of surface molecule TRAIL and FasL expression
Compared with the control group without significant difference (p > 0.05).
Embodiment 3.Nr-CWS promotes CD8+The expression of T cell intracellular granzyme B and perforin
CD8+T cell presses 1 × 106/ ml cell densities are inoculated with 96 orifice plates, per 100 μ l of hole.In 37 DEG C, 5%CO2Incubator
After middle culture 2 hours, the Nr-CWS for a concentration of 15 μ g/ml that experimental group addition culture solution dissolves, per 100 μ l of hole, if 3 are multiple
Hole.RPMI 1640 culture mediums are added in control group, per 100 μ l of hole, if 3 multiple holes.Isotype control group (i.e. negative control group) is made same
Sample processing.In 37 DEG C, 5%CO2Incubator in cultivate 24 hours and 48 hours.At last 4 hours of each detection time point,
Monensin solution is added per hole.Collect each group CD8+T cell marks anti-CD8a, anti-GrzB, anti-Prf stream respectively
Then formula fluorescence antibody washes away unbonded dyestuff, prepare the cell suspension of 500 μ l.Using flow cytometer detection CD8+T cell intracellular
The expression of granzyme B and perforin.Wherein, at least 10000 cells are collected using BD FACSDiva softwares.
As a result (being 24 hours see Fig. 3, wherein Fig. 3 A1 and Fig. 3 A2, Fig. 3 B1 and Fig. 3 B2 are 48 hours) display, Nr-CWS
Handle CD8+T cell 24 and after 48 hours, CD8+The expression of T cell intracellular granzyme B and perforin is notable compared with the control group
Increase (* p<0.05).
Embodiment 4.Nr-CWS promotes CD8+The expression of T cell TNF-α
CD8+T cell presses 1 × 106/ ml cell densities are inoculated with 96 orifice plates, per 100 μ l of hole.In 37 DEG C, 5%CO2Incubator
After middle culture 2 hours, the Nr-CWS for a concentration of 15 μ g/ml that experimental group addition culture solution dissolves, per 100 μ l of hole, if 3 are multiple
Hole.RPMI 1640 culture mediums are added in control group, per 100 μ l of hole, if 3 multiple holes.In 37 DEG C, 5%CO2Incubator in cultivate
24 hours.Nr-CWS groups and cellular control unit culture supernatant are collected, using the LEGENDplex of BioLegend companiesTM
Cytokine TNF-α, IL-2, IL-4, IL-5 in Mouse Th Cytokine Panel Kit detection cell culture supernatants
And IL-10.
As a result it is shown (see Fig. 4), Nr-CWS handles CD8+After T cell 24 hours, compared with the control group, TNF-α expresses water
HUD, which writes, increases (* p<0.05), IL-2, IL-4, IL-5 and IL-10 expression equal no significant difference (p > compared with the control group
0.05)。
More than, embodiments of the present invention are illustrated.But the present invention is not limited to the above embodiments.It is all
Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in the guarantor of the present invention
Within the scope of shield.
Claims (10)
1. Lyopgized Nocardia rubra-cell Wall Skeleton is being prepared for promoting CD8+Purposes in the drug of T cell proliferation.
2. CD8 of the Lyopgized Nocardia rubra-cell Wall Skeleton as non-treatment purpose+The purposes of T cell enhancer of proliferation.
3. a kind of external promotion CD8+The method of T cell proliferation, wherein give the CD8 of in vitro culture+T cell nocardia rubra
Cell wall skeleton.It is preferred that a concentration of 1-60 μ g/ml, the further preferred 2-30 μ g/ml of Lyopgized Nocardia rubra-cell Wall Skeleton,
More preferable 3-15 μ g/ml.
4. Lyopgized Nocardia rubra-cell Wall Skeleton is being prepared for promoting CD8+T cell function of surface molecule CD25, CD8+T cell
Function of surface molecule CD69, CD8+T cell intracellular granzyme B or CD8+Purposes in the drug of T cell intracellular perforin expression.
5. CD8 of the Lyopgized Nocardia rubra-cell Wall Skeleton as non-treatment purpose+T cell function of surface molecule CD25, CD8+T is thin
Cellular surface functional molecular CD69, CD8+T cell intracellular granzyme B or CD8+The purposes of T cell intracellular perforin expression accelerating agent.
6. a kind of external promotion CD8+T cell function of surface molecule CD25, CD8+T cell function of surface molecule CD69, CD8+T is thin
Born of the same parents' intracellular granzyme B or CD8+The method of T cell intracellular perforin expression, wherein give the CD8 of in vitro culture+T cell is red
Nocardial cell wall skeleton.It is preferred that a concentration of the 1-60 μ g/ml, further preferred 2- of Lyopgized Nocardia rubra-cell Wall Skeleton
30 μ g/ml, more preferable 3-15 μ g/ml.
7. Lyopgized Nocardia rubra-cell Wall Skeleton is being prepared for promoting CD8+Purposes in the drug of T cell TNF-α expression.
8. CD8 of the Lyopgized Nocardia rubra-cell Wall Skeleton as non-treatment purpose+T cell TNF-α expresses the purposes of accelerating agent.
9. a kind of external promotion CD8+The method of T cell TNF-α expression, wherein give the CD8 of in vitro culture+T cell red promise
Cattell bacterium cell wall skeleton.It is preferred that a concentration of 1-60 μ g/ml, further preferred 2-30 μ of Lyopgized Nocardia rubra-cell Wall Skeleton
G/ml, more preferable 3-15 μ g/ml.
10. purposes as claimed in any one of claims 1-9 wherein or method, wherein nocardia rubra Nr-8206.
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