CN103269705A

CN103269705A - Method for enhancing dendritic cell function by nocardia rubra cell wall skeleton

Info

Publication number: CN103269705A
Application number: CN2011800618259A
Authority: CN
Inventors: 张策; 张轶; 洪晓明; 张红阳; 张紫真
Original assignee: Liaoning Nakejia Biopharmaceutical Co ltd
Current assignee: Liaoning Nakejia Biopharmaceutical Co ltd
Priority date: 2010-12-23
Filing date: 2011-12-23
Publication date: 2013-08-28
Also published as: WO2012083884A1; CN102526119A

Abstract

The present invention provides a method for regulating mammal dendritic cell function by Nocardia Rubra cell wall skeleton and a use threreof.

Description

The method that Lyopgized Nocardia rubra-cell Wall Skeleton strengthens dendritic cell function

Lyopgized Nocardia rubra-cell Wall Skeleton strengthens the method and technology field of dendritic cell function

The present invention relates to immunotherapy field.Specifically, the present invention relates to the immunoregulatory method of Lyopgized Nocardia rubra-cell Wall Skeleton.Background technology

Lyopgized Nocardia rubra-cell Wall Skeleton effectively can transfer and activate the BMDC of body as a kind of immunomodulator, and BMDC is full-time antigen presenting cell in body immune system, is the key cells for starting body specific immunity.

1st, the effect of BMDC

BMDC is a critically important immune signal transmission cell in body immune system, the immune signal transmission effect of sole duty is undertaken in startup for specific immune system, τ cells are the important immunocytes that orientation removes pathogen in specific immunity, but the activation of T cell has to rely on the immune signal transmission effect of BMDC, so BMDC is the key cells in immune system, it is related to the success or failure that body specific immune function is effectively played.

In recent years, the sight of professional person's concern had turned to the research of human immune system in the industry, and people more focus on analysis the reason for for immunologic hypofunction, and then notice the phenomenon of the function reduction of BMDC, and such as BMDC is presented：Individual small, dendron is few, the low phenomenon of cell distribution density, it was recognized that it is the major reason of immunologic hypofunction that BMDC, which does not play a role,.

Therefore fall over each other to develop all kinds of dendritic cell vaccines both at home and abroad, be widely used in the function of enhancing BMDC, its design philosophy is：By the enhancing of dendritic cell function, and then start specific immune function.

Up to the present, clinically still without the immunomodulator for being preferably directed to dendritic cell function, the developmental research of every dendritic cell vaccine is also not over clinical test.

Therefore, effectively activation and to transfer the function of BMDC be still a common research topic in the industry, people expect that effectively can activate and transfer a new preparation birth for dendritic cell function.

2nd, clinically related indication

At present, clinically many diseases the reason for be exactly the immunologic hypofunction of body, and be immunized The low immunization of cell that all can not normally be played with immune system of function is relevant, and could not to play the immunization of cell anti-tumor effect that can not normally be brought into normal play with BMDC again relevant for immune system.

The generation of the most tumour of current human body, it is all relevant with body dendritic cell function decrease, due to the reduction of dendritic cell function so that the specific immune function of body is unable to effectively start.

Because the reduction of dendritic cell function so that the signal of pathogen can not effectively pass to τ cells by BMDC, therefore cause T cell to be activated and play a role.

And internal tumour relies primarily on the scavenging action of τ lymphocytes, the development of tumour could be effectively controlled.Therefore, it can be said that clinically whether normal the function of the BMDC in the occurrence and development of tumour, with the immune system of body is relevant.

So, clinically occur and control the key of tumor development about pre- preventing tumor, can exactly there is method to transfer and activate the function of BMDC, this has become focus problem clinically.

3rd, present situation clinically

Immunization therapy for tumour is the 4th kind is treated tumour after operation, radiotherapy, chemotherapy new method.The immunization therapy of tumour is current study hotspot, BMDC is relevant with the occurrence and development of tumour, if dendritic cell function defect or low in patient's body, can not effective submission tumour antigen, specific immunity T cell can not be activated, then cause immunity of organism incompetent and immune tolerance so that tumour is able to occurrence and development.

The immature state of BMDC, it is impossible to effectively offer antigen, as can effectively start specific immunity key link.At present, people are mainly using the method for exploitation dendritic cell vaccine, to solve the problem of dendritic cell function is weakened.

Many examples are proved, are played a leading role by the T cell of dendritic cell ciita during antineoplastic immune.Immunization therapy based on BMDC turns into one of Main Means of following antineoplaston in the future.

The mainstream research direction of this area：It is to find to use in vivo and in vitro, compatibility is good, safer and efficient antitumor dendritic cell vaccine mode.Contrasted with the situation of burning hot research and development vaccine；It can also be used for transferring and activating BMDC without specific medicine.

4th, the problem of dendritic cell vaccine is applied and existed

Tumor vaccine based on BMDC turns into new strategy and the master of immunotherapy of tumors Offense to.By loaded in vitro BMDC, vivo immunization therapeutic strategy is fed back；The strategy of internal primed dendritic shape cell receives many curative effects, but also run into many problems in dendritic cell vaccine application：

1., in vitro culture is easy to pollution；2., in vitro culture cost is high；3. autoimmune disease, is easily triggered；4., drug effect is unstable；5., wait to find optimal preparation method；6., continue to screen the mode that preferable tumour antigen is loaded；7., technical difficulty is larger；8., large-scale culture prepares difficult；9. clinical treatment otherness is big；, knurl seedling feed back attention avoid τ cell depletions.

Dendritic cell tumor vaccine has shown that out wide potential applicability in clinical practice, it will be a kind of antitumor strategy of very promising and use value.But simultaneously it should also be recognized that, the Dendritic Cell Vaccines in Tumor Immunotherapy of current research is far from reaching the effect that people expect, clinical experimental stage is still in, mainly many important molecular immune mechanism such as treat immunologic escape, immune tolerance, cytotoxic T cell（CTL) activity reduction or reactionless, and is affected by the surrounding environment during BMDC present antigen, the problems such as need to be studied and illustrate.

In addition, there is presently no generally acknowledged answer for the problems such as how finding the optimal BMDC optimal maturing dendritic cell of method choice prepared and the state activated and the method for the load of tumour antigen.

In a word, dendritic cell vaccine not yet reaches the desirable of people, still in clinical experimental stage, and the research and development of dendritic cell vaccine still belong to crudity.The content of the invention

Tested by multiple bioequivalence, inventor has found effectively transfer and dendritic cell activated as the Lyopgized Nocardia rubra-cell Wall Skeleton of biological agent, BMDC is full-time presenting cell in body immune system, and can BMDC activate extremely important for starting body specific immunity.Therefore this experimental result can illustrate：Lyopgized Nocardia rubra-cell Wall Skeleton is conducive to BMDC to find and recognize pathogenic antigens and be acted upon by activating and transferring BMDC, therefore is more conducive to activation τ cells, makes the specific immunity of human body be easier to effectively be activated.

The experimental result explanation of the present invention, Lyopgized Nocardia rubra-cell Wall Skeleton can substantially increase the density of BMDC and the form and function of rapid improvement BMDC, be active BMDC. After Lyopgized Nocardia rubra-cell Wall Skeleton effect on human body, it can effectively activate and strengthen the function of BMDC, and then beneficial to transfer human body specific immune function, so that τ cells are easy to be activated, promote the activation of human body cell immune system, cause human body specific immunity anti-tumor immune response.

Lyopgized Nocardia rubra-cell Wall Skeleton starts patient's autospecific tumor-killing mechanism by correcting body immune deficiency, and tumour cell is removed by the immunologic function of itself is activated.

The discovery of Lyopgized Nocardia rubra-cell Wall Skeleton this dendritic cell activated effect, filled up clinically does not have the blank of safe and effective medicine for the low symptom of dendritic cell function, finish the difficult situation that doctors there is no good plan in face of the low patient of numerous dendritic cell functions, people there are new treatment means for the illness of immunocompromised.

Therefore, one aspect of the present invention is related to Lyopgized Nocardia rubra-cell Wall Skeleton and is preparing the purposes in being used to adjust the immune formulation of mammalian dendritic shape cell function, wherein described regulation includes Lyopgized Nocardia rubra-cell Wall Skeleton activation, transfers and strengthens the function of mammalian organism BMDC.

Mammal of the present invention includes people.

Many diseases known in the art are lowly relevant with dendritic cell function, for example, the low illness of the dendritic cell function treated by Lyopgized Nocardia rubra-cell Wall Skeleton of the present invention.

In purposes of the present invention, BMDC can be Langerhans cell.

In purposes of the present invention, can as needed be determined by those skilled in the art using the effective dose of immune formulation, such as the dosage of required immune formulation can be 5ug-1200ug.

In purposes of the present invention, immune formulation can be administered by internal, subcutaneous or route of exposure, be preferably to be administered with skin and/mucosal contact.

In purposes of the present invention, immune formulation can include pharmaceutically acceptable carrier or excipient.Any appropriate carrier known to those skilled in the art or excipient can be used in the present invention.Preferably, workable excipient includes freeze-dried formulation, cream form or liniment.

The invention further relates to a kind of method for adjusting mammalian dendritic shape cell function, Lyopgized Nocardia rubra-cell Wall Skeleton including directly using from effective dose to mammal, wherein described regulation includes Lyopgized Nocardia rubra-cell Wall Skeleton activation, transfers and strengthens the function of mammalian organism BMDC.

Therefore, present disclosure is related to following aspects：

1st, by the acting body of Lyopgized Nocardia rubra-cell Wall Skeleton and skin and mucous membrane, So that body BMDC is activated comprehensively, and then the immunologic function of body is set greatly to strengthen；

2nd, the enhancing of dendritic cell function is embodied in, the increase of the density of BMDC, and the morphologic change of BMDC is into state of activation；

3rd, the enhancing of dendritic cell function is passed through so that the startup of the specific immune function of body has good basis；

4th, in Lyopgized Nocardia rubra-cell Wall Skeleton effect body processes, medicine is Gamma Magnitude, safe and reliable to have an effect rapidly；

The present invention has following features：

1st, after Lyopgized Nocardia rubra-cell Wall Skeleton micro constitutent effect body, rapid after several hours BMDC can be effectively activated, drug effect is swift in response after medication；

2nd, in the presence of Lyopgized Nocardia rubra-cell Wall Skeleton micro constitutent, the density of the BMDC of body substantially increases, and the expansion of BMDC form is full, and BMDC can fully be activated；

3rd, in the appropriate composition effect body processes of Lyopgized Nocardia rubra-cell Wall Skeleton, body has no toxic side effect, and shows good security；

4th, clinically application operating is easy for Lyopgized Nocardia rubra-cell Wall Skeleton, facilitates patient to use.Brief description of the drawings

Fig. 1:Langerhans cell colored graph.A is 288x, and b is 1152 χ.

Fig. 2:Mouse and dextran control group mice Langerhans cell figure is administered in Na Kejia groups.Fig. 3:The influence line chart of Na Kejia groups and dextran group to mouse ear LC cells.Fig. 4:The expression of S100, CDla, HLA-DR after normal person's cervical biopsy tissue, cervicitis biopsy, cervicitis Na Kejia treatments in biopsy.Embodiment

Test material

1. medicine：Lyopgized Nocardia rubra-cell Wall Skeleton（Na Kejia）, source：Liaoning Nakejia Biological Pharmaceutical Co., Ltd., authentication code：Chinese medicines quasi-word S20030009.

2. animal：68 weeks Healthy female BALB/c mouses, SPF grades, 14, body weight (20 ± 2>g.Source：Cloud water chestnut animal breeds factory in Beijing. 3. numbering Ζ Μ -0136 of mouse anti-human HLA-DR, 250 μ 1, the production of Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge.

4. mouse ABC kits：L/4kit, Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge（VECTOR companies of the U.S.）.

5. rabbit-anti people HLA-DR (RabbitAnti-HLA-DR), 0.2ml, numbering BS-1198R, Beijing Bo Aosen Bioisystech Co., Ltd.

6. rabbit immunohistochemical staining kit：3ml, numbering SP-0023, Beijing Bo Aosen Bioisystech Co., Ltd.

7. rat anti-human HLA-DR, 0.25ml, numbering MCA71R, serotec companies produce.

8. biotinylation rabbit-anti rat IgG (H+L), numbering ZB-2040, the production of Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge.

9. AEC zymolyte kits:2ml, numbering zli-9036, lot number K106718A, the term of validity 08/2011, Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge.

10. PBS：2000ml/ bags, Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge.

11. GVA mountants：5ml, numbering zli-9051, lot number P92304H, the term of validity 07/2011, Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge.

Experimental design

The checking thinking of the present invention is the principle using bioequivalence, by the way of zoopery, inventor have selected the experimental method in Chinese Medical Sciences University's state key subject laboratory, pass through multiple multigroup mouse experiment in vivo, the calculating with the statistical calculation method of science is detected by advanced detection means, it was demonstrated that the efficiency of BMDC can be transferred and activated to Lyopgized Nocardia rubra-cell Wall Skeleton.

Animal protocol and step

The thinking and design principle of experimental program：

Bioequivalence experiment is carried out using small white mouse, several experimental groups and two control groups are divided into immediately, with reference to the related experimental methods of professional paper, the characteristics of being tested with reference to inventor itself formulates the animal protocols of the checking present invention.

Embodiment 1

Test procedure

1. packet：3 mouse are taken to be randomly divided into three groups and number, i.e. blank group（No. 1）、 20μ₈Na Kejia groups (No. 2:) and dextran excipient control group（No. 3）.No. 1, No. 3 uses primary antibody And secondary antibody（Rat anti-human HLA-DR), No. 2 primary antibodies are using rabbit-anti people HLA-DR.

2. operating procedure：

Naive mice 1 is taken, is operated according to the method that the true epidermis of mouse is separated, corium and epidermis are separated.No. 2 and No. 3 mouse inject respectively 0.2ml receive can good solution and 0.2ml Dextran 40 solution, carried out cutting ear separation in second day, and carry out immunohistochemical staining, microscopy.

The true epidermis separation of mouse：

1. etherization mouse, cuts a part for ear.

2. the sebum and scurf of blade scraper surface are used, PBS is rinsed 3-5 times repeatedly.

3. the skin of mouse ear is separated with tweezers.Blade strikes off cartilage, and is cut into 0.5cmx0.5 cm fritters.

4. EDTA separating liquids are added（PH7.2 skin graft is taken out in) after 37 °C of water-baths, 80min to separate true epidermis, stays epidermis standby.

3. antibody labeling：

1. acetone room temperature fixes 4 °C of epidermis, and 20min, PBS rinses 5 min, 2 times；

2. epidermis is put into containing 1：Shaken in the microcentrifugal tube of 100 rat anti-human HLA-DR (No. 2 are rabbit-anti people HLA-DR) antibody, 4 °C overnight；

3. epidermis is taken out, PBS rinses 5 min, 2 times, is put into containing 1:In the microcentrifugal tube of rabbit-anti rat IgG (No. 2 are goat anti-rabbit igg) antibody of 50 bioid, it is incubated after 1.5h, PBS rinses 5min, 2 times；

4. epidermis is put into plus horseradish enzyme is marked in chain enzyme avidin microcentrifugal tube, is incubated 60min, and PBS rinses 5min, 2 times.

4. immunohistochemical staining（Used according to AEC kits）

Before dyeing, each 50 μ 1 of No. 123 reagents is added to the microcentrifugal tube for having filled 1ml distilled water respectively, is kept in dark place, in 30min effectively.

1. at room temperature, Ι Ο Ο μ Ι dyeing working solution carefully is added in slide, is put into epidermis（Corium is upwardly）, it is immersed in working solution.

2. 10 are incubated at room temperature to 20min, or until there is red in sample.

3. supracutaneous dyeing working solution is removed.

4. at room temperature, slide is inserted in the PBS cushioning liquid that 50ml is provided for oneself and soaks 5s.

5. supracutaneous PBS cushioning liquid is removed.

6. glycerin gelatine mounting after air-drying. 7. observe under an optical microscope at once：The positive presents red.

Embodiment 2

Test procedure

1. packet：8 mouse are taken to be randomly divided into two groups and number, i.e. 40 μ₈Na Kejia groups (No. 1-6:) and dextran excipient control group（No. 1-2）.

2. operating procedure：

(^g/ml Na Kejia solution, dextran control group injects 0.2ml3% Dextran 40 solution to Na Kejia groups injection 0.2ml4, is specifically administered and takes ear dyeing time see the table below：

The true epidermis separation of mouse：

1. etherization mouse, cuts a part for ear.

3. antibody labeling：

1. acetone room temperature fixes 4 °C of epidermis, and 20min. PBS rinse 5 min, 2 times；

2. epidermis is put into containing 1:In the microcentrifugal tube of 100 rat anti-human's HLA-DR antibody, 4 °C overnight；

3. epidermis is taken out, PBS rinses 5 min, 2 times, is put into containing 1:In the microcentrifugal tube of the rabbit-anti anti-rat IgG antibody of 50 bioid, it is incubated after 1.5h, PBS rinses 5min, 2 times；

4. immunohistochemical staining（Used according to AEC kits） Before dyeing, each 50 μ 1 of 12 No. 3 reagents is added to the microcentrifugal tube for having filled lml distilled water respectively, is kept in dark place, in 30min effectively.

1. at room temperature, Ι Ο Ο μ Ι dyeing working solution is added in slide, epidermis is put into（Corium is upwardly）, it is immersed in working solution.

3. supracutaneous dyeing working solution is removed.

4. at room temperature, slide is inserted in the PBS cushioning liquid that 50ml is provided for oneself and soaks 5s

5. supracutaneous PBS cushioning liquid is removed.

6. glycerin gelatine mounting after air-drying.

7. observe under an optical microscope at once：The positive presents red.

Observation index and criterion

1. observation index：Randomly select 10 continuous visuals field to each sample to count, LC is determined to see cell space and dendron simultaneously.Positive LC density (individual/mm²>=positive cell number/epidermis area.

2. staining power judges：Dye-free be negative slight stain be weakly positive dyeing it is shallower be positive staining relatively depth be moderate positive dyeing depth be strong positive (+++).

3. statistical method：LC density ratios are relatively examined using paired t.

Result of the test

The antibody that embodiment 1 is produced using Serotec companies is dyed, and No. 1 piece is shown in many eucaryotic cell structures for dying salmon pink under mirror, through being determined as Langerhans cell（Fig. 1 a figures are that 288 χ b figures are 1152 χ), illustrate to dye successfully.

Embodiment 2 is tentatively studied the effect of mouse ear Langerhans cell Na Kejia.Through overtesting, each group mouse Langerhans cell uniform dyeing, each group picture is shown in Fig. 2 (picture is 288x).Fig. 3 shows the influence line chart of Na Kejia groups and dextran group to mouse ear LC cells.The density of table 1, mouse ear Langerhans cell（T is examined）It see the table below:

No. 3 815.0 695.0 995.0 875.0 890.0 854.0 ± 110.0

No. 4 883.0 853.0 857.0 886.0 867.0 869.2 ± 14.9

No. 5 750.0 730.0 857.0 730.0 813.0 776.0 ± 56.7

No. 6 917.0 833.0 927.0 833.0 833.0 868.6 ± 48.9 dextrose 1,604.2 604.2 704.8 648.2 591.6 630.6 ± 46.7 acid anhydride groups No. 2 667.1 742.6 944.0 585.3 755.2 738.8 ± 133.3,

In embodiment 1：The orange-red structure that Microscopic observation is arrived, is defined as Langerhans cell.

In example 2：Understood by the analysis to test structure：

1 can see from the contrast of picture, Na Kejia groups administration mouse（2nd, 3,4,5, No. 6 mouse）Ear's Langerhans cell is significantly better than non-administration mouse (No. 1 mouse in quantity and cellular morphology）And higher than dextran control group.

2 can see from density t check tables, Na Kejia groups administration mouse（2nd, 3,4,5, No. 6 mouse）Ear's Langerhans cell density is obviously higher than non-administration mouse（No. 1 mouse）, while also above dextran control group.

3 can see from the line chart of density, in general, and Na Kejia groups administration mouse LC cell densities do not have significant change with the increase of administration number of times.

By above Several Analysis, illustrate to receive can Canon enough promote mouse ear Langerhans cell quantity increase, while Langerhans cell becomes big, full, dendron quantity increase, illustrate that Na Kejia has activation to the function of Langerhans cell.

1st, statistics explanation：Lyopgized Nocardia rubra-cell Wall Skeleton dramatically increases the density of BMDC；

2nd, caption is contrasted：Lyopgized Nocardia rubra-cell Wall Skeleton is obviously improved the form of BMDC；

3rd, comprehensive conclusion：Lyopgized Nocardia rubra-cell Wall Skeleton effective activation and BMDC is transferred.

Humidification of the Lyopgized Nocardia rubra-cell Wall Skeleton of embodiment 3 in cervical lesionses to BMDC

From in April, 2011 in by the end of November, 2011, through under informed consent, randomly selecting 73 clients of Beijing Wangjing hospital, the wherein normal person of uterine neck 58, virus in patients with cervicitis 15.To 6 in virus in patients with cervicitis using marketed drugs " Na Kejia "（Known nocardia rubra is thin Cell wall backbone composition, Liaoning Nakejia Biological Pharmaceutical Co., Ltd.'s production）Treatment, remaining 9 virus in patients with cervicitis is not as treating control group.Treatment method is：Added medicine in outpatient service in 2-3 days after clean period, physiological saline thoroughly cleaning vagina and uterine neck after conventional vulvar disinfection, take Na Kejia 120 to soak magnetic tape trailer cotton balls after being diluted with the ml of physiological saline 4, wipe again and uterine neck is placed on after uterine neck and is taken out after retaining 24 hours.The next day once, 10 times are 1 course for the treatment of, and menstrual onset is discontinued, and are discontinued after the full course for the treatment of of menopause person's medication.Take pathological tissue standby after 10 days.

58, normal person's cervical biopsy tissue is taken, cervicitis biopsy 9, biopsy 6 after cervicitis Na Kejia treatments is respectively adopted immunohistochemical method and taken cervical biopsy tissue is observed and analyzed.

Materials and methods

Clinical human cervical cancer 1 tissue sampling：

Biopsy specimen fixes 24h, specimens paraffin embedding slices through 4% formaldehyde.

Clinical Na Kejia treatment patient uterine's necks biopsy materials time point is that 10 days after medication, each biopsy provides 3x5mm biopsy blocks, and tissue block is fixed for 10% paraformaldehyde, FFPE, section.

Main agents：

Specific primary antibodies：

(1) distribution and quantitative analysis of anti-human CDla, S100, HLA-DR monoclonal antibody the observation mature dendritic cell of mouse and immature dendritic cell.

(2) the anti-human CD56 monoclonal antibodies of mouse, CD56 is a kind of natural adhesion molecule, and normal expression is in NK cells（Natural killer cells) and T cell subgroup, observe the distribution and quantitative analysis of Ν Κ cells.

(3) anti-human CD3, CD4, CD8 monoclonal antibody of mouse, observes the development of T cell and CD4/CD8 ratio, declines and represent immune function depression.

Secondary antibody：Biotinylated derivative, goat anti-mouse igg.

Instant UltraSensitivdl Ms-P immunohistochemical kits.

DAB chromogenic enzyme substrate agent.

Other reagents：

(1) PBS buffer solutions（74) 0.01Mol/L, ρ Η 72 prepares： NaCl 8.5g, Na₂HP0₄ 1.07g, KH₂PO₄0.39g.Three of the above composition is first dissolved in 100ml distilled water, after molten, fills up distilled water to 1000ml. (2) citrate buffer（0.01Mol/L, pH6.0) prepare：Trisodium citrate 3g, citric acid 0.4g are weighed respectively, appropriate distilled water is added, and distilled water is supplemented after being sufficiently mixed to 1000ml

(3) edta buffer liquid（0.5Mol/L, pH8.0) prepare：186.1g EDTA-2H are dissolved in 700ml water₂0, adjust pH to 8.0 with lO Mol/L NaOH, add water to 1000ml.

(4) TBS buffer solutions（1.0 Mol/L, pH8.0) prepare：121g Tris alkali is dissolved in 800ml water, adjusts pH to 8.0 with IN HC1, adds water to 1000ml.

(5) 3% methanol -0₂Solution is prepared：Use 30% H₂0₂Prepared with 80% methanol solution.

(6) envelope mounts agent：Glycerine and carbonate buffer solution（0.5 mMol/L, pH9.0 9.5) mixed in equal amounts.

(7) 0.3 %H₂O₂, DAB chromogenic enzyme substrate agent, haematoxylin, graded ethanol, L- poly-D-lysines, Normal Goat Serum, distilled water, dimethylbenzene etc..

The reagents such as above antibody are purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge.

Laboratory apparatus：

(1) light microscope and camera

(2) refrigerator

(3) it is incubated wet box

(4) micro-wave oven

(5) water isolation type electro-heating standing-temperature cultivator

(6) water bath, high-temperature resistance plastice slide holding frame, adjustable micropipettor, PAP draw a circle pen, timer, slide etc..

Experimental procedure：

1. immunohistochemical staining：Take biopsy after normal person's cervical biopsy tissue or cervicitis biopsy or cervicitis Na Kejia treatments, 4% formaldehyde is fixed, routine paraffin wax is embedded, cut 5 μ η ι thickness sections, it is attached on the slide for scribbling poly-D-lysine, inserts in 60 °C of baking ovens and toast 20 minutes.

2. take biopsy dimethylbenzene after the thick normal person's cervical biopsy tissues of 5 μ η ι or cervicitis biopsy or cervicitis Na Kejia treatments to dewax, decreasing gradient alcohol aquation.

3. histotomy is washed 23 times with PBS, after each 5 minutes, 3 % 0 of drop power mouthful₂- 80% methyl alcohol mixed liquor, is stored at room temperature 10 minutes, to remove endogenous peroxidase activity.

4. hot high pressure reparation：Histotomy is washed 23 times with PBS, after each 5 minutes, in boiling EDTA buffer solutions are added in water（0.5Mol/L,) or citrate buffer (O.OlMol/L pH8.0, pH6.0), the lid of stainless steel pressure cooker is covered, but without locking, slide is placed on metallochromy frame, slow pressurization, slide is set to be soaked in buffer solution 5 minutes, then by cover lock, minor valve, which will rise, to be come.After 10 minutes, thermal source is removed, inserts in cold water, lid is opened after minor valve is sunk.

5. histotomy is washed 23 times with PBS, after each 5 minutes, Normal Goat Serum confining liquid, room temperature 20 minutes is added dropwise.Surplus liquid is got rid of, is not washed.

6. different first antibodies are added dropwise respectively（CDla, S100, HLA-DR, CD3, CD4, CD8 and CD56) each 50 μ 1, be stored at room temperature 1 hour or 4 °C overnight or 37 °C 1 hour.

7. 4 °C overnight after need to be 45 minutes in 37 °C of rewarmings.

8. PBS is washed 3 times, each 5 minutes, 1,37 °C of 40 50 μ of secondary antibody is added dropwise and stands 1 hour.

9. PBS wash 3 times it is each 5 minutes. 0.3 %H₂O₂, DAB chromogenic enzyme substrate agent monitors under light microscopic, develops the color at room temperature, sucks nitrite ion at once during thin background color whne cell color, distilled water rushes rapidly the addition in good time terminating reactions of PBS after three times.

10. PBS or running water, are rinsed 10 minutes, haematoxylin is redyed 30 seconds, up dehydration of alcohol（70%th, 80%, 90% alcohol, 5 minutes X 1 time, 95 % alcohol 5 minutes χ 2 times, 100% χ 3 times of alcohol 5 minutes）, dimethylbenzene is transparent, 50% neutral gum mounting.

Experiment contrast：

Experiment is equipped with positive, negative control every time, makees positive control with itself known positive section, primary antibody is replaced with PBS as negative control

As a result criterion：

Cell count uses x400 light Microscopic observations.

Staining power is observed using Olympus UPlan FL x40 object lens：The cell of the dyeing of display cell membrane or cytoplasm dyeing is included in Positive Cell Counts.

1. CDla, S100, HLA-DR are display prematurity dendritic cells specific antibody, with cell membrane or cytoplasm in yellowish-brown under light microscopic, and the cell with the irregular projection of dendroid is the positive.

2. CD3, CD4, CD8 are respectively display Th cells, Tc cells and Ts cell-specific antibodies, contain pale brown particle as the positive using in cytoplasm under light microscopic.

3. CD56 is display NK specific antibody, counted under light microscopic with cell membrane and nucleus in brown color tinter. Experimental result：

1st, the observation analysis of S100, CD la, HLA-DR cell expression

Normal person's cervical biopsy tissue：S100, HLA-DR react in weakly positive, and CDla is in weakly positive or feminine gender.

Cervicitis biopsy：S100, HLA-DR are in that, compared with strong positive reaction, and CDla is positive or negative.

Biopsy after cervicitis Na Kejia treatments：S100, HLA-DR react in weakly positive, and CDla is in weakly positive or feminine gender.

Result and Fig. 4 more than, in inflammatory tissue, BMDC has born stronger immunosurveillance and has presented the function of antigen, and function is more active.After being treated through Na Kejia, the normal immune surveillance function state of BMDC has been recovered due to enhancing the function of BMDC.

2nd, the observation analysis of CD56 (NCAM) cell expression

Normal person's cervical biopsy tissue：CD56 is positive or negative.

Cervicitis biopsy：CD56 is negative.

Biopsy after cervicitis Na Kejia treatments：CD56 is visible to be positive or negative.As a result show, NK cells take part in immunologic function after normal and cervicitis Na Kejia treatments in biopsy, and by the enhancing of dendritic cell function, have activated the function of T cell.

3rd, CD3, CD4, the observation analysis of cd8 cell expression

Normal person's cervical biopsy tissue：CD3, CD4, CD8 are positive or negative.

Cervicitis biopsy：CD3, CD4 are negative.A small number of visible a small amount of CD8 positive cells of case.

Biopsy after cervicitis Na Kejia treatments：CD3, CD4, CD8 recover to be positive or negative result.

As a result show, under inflammatory conditions, T cell is in the state of protection body, it was observed that inflammation part can see the T cell largely assembled.T cell has recovered normal distribution again in biopsy after Na Kejia treatments. Send out τ cells and occur specific immune response.Nocardia rubra cell skeleton has effects that to activate and strengthen BMDC effect in cervical lesionses.

Claims

Claims:

1st, Lyopgized Nocardia rubra-cell Wall Skeleton is preparing the purposes in being used to adjust the immune formulation of mammalian dendritic shape cell function.

2nd, purposes according to claim 1, wherein described regulation includes Lyopgized Nocardia rubra-cell Wall Skeleton activation, transfers and strengthens the function of mammalian organism BMDC.

3rd, purposes according to claim 1 or 2, it is characterised in that described BMDC includes Langerhans cell.

4th, the purposes according to any one of claim 1-3, it is characterised in that the dosage of described immune formulation is 5ug-1200ug.

5th, the purposes according to any one of claim 1-4, it is characterised in that described immune formulation is administered by internal, subcutaneous or route of exposure.

6th, the purposes according to any one of claim 1-4, it is characterised in that described immune formulation includes pharmaceutically acceptable carrier or excipient.

7th, purposes according to claim 6, it is characterised in that described excipient includes freeze-dried formulation, cream form or liniment.

8th, the method for adjusting mammalian dendritic shape cell function, including to mammal directly using the Lyopgized Nocardia rubra-cell Wall Skeleton of effective dose.

9th, method according to claim 8, wherein described regulation includes Lyopgized Nocardia rubra-cell Wall Skeleton activation, transfers and strengthens the function of mammalian organism BMDC.

10th, method according to claim 8 or claim 9, it is characterised in that described BMDC includes Langerhans cell.