CN114848667A

CN114848667A - New application and preparation method of hyaluronic acid fragment

Info

Publication number: CN114848667A
Application number: CN202110270013.0A
Authority: CN
Inventors: 郭田田; 王家麒; 李鑫荣; 贾潇潇; 吴书音; 惠鑫瑶; 桐辉; 崔家友; 张之辉; 丛振昱; 王起飞; 王凤舞; 宋琳; 惠觅宙
Original assignee: Qingdao Huinuode Biotechnology Co ltd; Shaoxing Huihui Technology Co ltd; Qingdao Agricultural University
Current assignee: Qingdao Huinuode Biotechnology Co ltd; Shaoxing Huihui Technology Co ltd; Qingdao Agricultural University
Priority date: 2020-03-12
Filing date: 2021-03-12
Publication date: 2022-08-05
Also published as: CN111249302A

Abstract

The invention discloses an application of a hyaluronic acid fragment as a human monocyte migration promoter, an application of the hyaluronic acid fragment as an immunomodulator of human lymphocytes and liquid reflux and lymphocytes through subcutaneous or intravenous injection, and an application of the hyaluronic acid fragment as a human neutrophil migration inhibitor; the hyaluronic acid fragment is prepared by sufficiently hydrolyzing hyaluronic acid raw material with PH20, can pass through 0.22 μm filter membrane, and has average molecular weight of 35 + -8 KDa. The invention also discloses application of the hyaluronic acid fragment in preparing a medicament for treating inflammatory diseases related to human monocyte and neutrophil migration and application in preparing a medicament for treating inflammatory diseases related to human lymphocyte and liquid reflux. The invention also discloses a method for preparing the hyaluronic acid fragments and a method for preparing the hyaluronic acid fragments with different molecular weights. The invention discovers a new preparation method, an action mechanism and application of the hyaluronic acid fragment, and lays a foundation for pharmaceutical research of the hyaluronic acid fragment.

Description

New application and preparation method of hyaluronic acid fragment

Technical Field

The invention relates to the field of biomedicine, in particular to a new application and a manufacturing method of a hyaluronic acid fragment.

Background

At present, the scientific society has no unified opinion on the physiological function and the pharmaceutical efficacy of the human extracellular matrix hyaluronic acid. The earliest hyaluronic acid products were all arthritis-treating injections with an average molecular weight of more than 1200kDa, such as Durolane and Synvisc. Literature studies indicate that the physiological and pharmaceutical efficacy of hyaluronic acid is related to its molecular weight size. Literature studies have also shown that hyaluronic acid fragments of average molecular weight 35kDa extracted from human colostrum have a variety of physiological and pharmaceutical effects. Hyaluronic acid fragments with an average molecular weight of 35kDa extracted from human colostrum were produced by hyaluronidase cleavage in female breasts. The female breast is the gonad and its hyaluronidase is the sperm acrosome hyaluronidase PH20 of the male gonad testis.

The inventor uses recombinant human hyaluronidase PH206 hours to enzymolyze hyaluronic acid raw material with molecular weight of 300-1600kDa to manufacture bioactive hyaluronic acid fragment B-HA with average molecular weight of 35 kDa. The present inventors also produced class 1 Medical Device B-HA products (LUQIN Food Drug Medical Device registration number:20190021) using bioactive hyaluronic acid fragments B-HA as starting materials. These class 1 medical device B-HA products have been used for out-of-the-specification clinical studies to clinically manifest red, swollen, hard and painful mucocutaneous inflammation. The clinical research results of the external application of the skin mucosa show that the bioactive hyaluronic acid fragment B-HA HAs the activity of definitely inhibiting the clinical red swelling and pain of the skin mucosa), provides practical basis for the clinical anti-inflammatory activity of the skin mucosa, and the clinical research results are published and patented in local magazines.

The prior art HAs limited research on stable manufacturing method, manufacturing principle, action mechanism of bioactivity, new bioactivity and potential new clinical application of bioactive hyaluronic acid fragment B-HA, and further research and development are needed.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a new application of the hyaluronic acid fragment prepared by a special preparation method and a preparation method thereof.

In order to solve the technical problems, the invention mainly uses hyaluronidase PH20 to sufficiently or slightly excessively and sufficiently hydrolyze the high or medium molecular weight hyaluronic acid raw material, and researches a stable manufacturing method and a manufacturing principle for manufacturing the hyaluronic acid fragment B-HA with the average molecular weight of 35 +/-8 KDa. The invention also researches the action mechanism and potential new application of the biological activity of the hyaluronic acid fragment B-HA with the average molecular weight of 35 +/-8 KDa.

Based on the research, the following technical scheme is obtained:

in one aspect, the present invention provides a novel use of a hyaluronic acid fragment, the hyaluronic acid fragment being: hyaluronic acid fragments with average molecular weight of 35 + -8 KDa, which can pass through 0.22 μm (220nm) pore size filter membrane, are prepared by using recombinant human hyaluronidase PH20 or extracted bovine hyaluronidase PH20, and sufficiently or slightly excessive enzymolysis of high or medium molecular weight hyaluronic acid raw material; the application is as follows: use of the hyaluronic acid fragments as a human monocyte migration-promoting agent; and/or the use of said fragments of hyaluronic acid as immunomodulators of human lymphocytes and fluid reflux and lymphocytes by subcutaneous or intravenous injection; and/or the use of said hyaluronic acid fragments as inhibitors of human neutrophil removal.

In another aspect, the present invention also provides a novel use of a hyaluronic acid fragment, wherein the hyaluronic acid fragment is: hyaluronic acid fragments with average molecular weight of 35 + -8 KDa, which can pass through 0.22 μm (220nm) pore size filter membrane, are prepared by using recombinant human hyaluronidase PH20 or extracted bovine hyaluronidase PH20, and sufficiently or slightly excessive enzymolysis of high or medium molecular weight hyaluronic acid raw material; the application is as follows: the application of the hyaluronic acid fragment in preparing a medicament for treating inflammatory diseases related to human monocyte migration, or preparing a human monocyte migration promoter; and/or, the hyaluronic acid fragment is used for preparing a medicament which is injected subcutaneously or intravenously and is used for treating inflammatory diseases related to human lymphocytes and liquid reflux, or is used for preparing an immunomodulator which is injected subcutaneously or intravenously and is used for treating human lymphocytes and liquid reflux and lymphocytes; and/or, the application of the hyaluronic acid fragment in preparing a medicament for treating inflammatory diseases related to human neutrophil removal, or preparing an inhibitor for treating human neutrophil removal.

Further, the human mononuclear cells (human freshly extracted mononuclear cells) are mainly lymphocytes, and may also include a small amount of macrophage precursor mononuclear cells; the hyaluronic acid fragments inhibited human neutrophil removal at higher concentrations (150-.

In still another aspect, the present invention provides a method for producing a hyaluronic acid fragment, comprising the step of enzymatically hydrolyzing a high or medium molecular weight hyaluronic acid material with a sufficient or slight excess amount to produce a hyaluronic acid fragment having an average molecular weight of 35 ± 8KDa using recombinant human hyaluronidase PH20 or extracted bovine hyaluronidase PH 20.

Further, the fragments can be passed through a 0.22 μm (220nm) pore size filter; the enzymolysis time is 2-6 hours.

Further, a high or medium molecular weight hyaluronic acid injection is mixed with the recombinant human hyaluronidase PH20 or the extracted bovine hyaluronidase PH20 injection, and the hyaluronic acid fragment injection with the average molecular weight of 35 +/-8 KDa is prepared by enzymolysis.

Further, the enzymolysis time is 10-20 minutes.

Further, the sufficient or slight excess sufficient enzymatic hydrolysis is: (1) the molecular weight of the hyaluronic acid fragments prepared by sufficiently or slightly excessively performing enzymolysis on the neutralized high molecular weight hyaluronic acid raw material for 10-20 minutes is basically consistent with that of the hyaluronic acid fragments prepared by sufficiently or slightly excessively performing enzymolysis on the neutralized high molecular weight hyaluronic acid raw material for 1-6 hours, and the coefficient of variation CV is less than 15%; (2) the low molecular hyaluronic acid fragment product of which more than 99 percent of high or medium molecular hyaluronic acid raw material is sufficiently enzymolyzed by enough or slight excess, is completely and smoothly filtered by a 0.22um pore size filter membrane; (3) the hyaluronidase activity has no residue or little residue after sufficient or slight excess and sufficient enzymolysis reaction, the residue is less than 15 percent, and the hyaluronidase can be completely inactivated in 45 minutes at 80 ℃.

In another aspect, the present invention provides a method for producing hyaluronic acid fragments with different molecular weights, the method comprising: recombinant hyaluronidases with different molecular weights or extracted hyaluronidases are used for carrying out enzymolysis and cutting on high molecular weight hyaluronic acid raw materials to prepare hyaluronic acid fragments with different molecular weights.

Further, the recombinant hyaluronidase or extracted hyaluronidase with different molecular weights is: full-length, partial and fused recombinant hyaluronidases or extracted hyaluronidases of a variety of different species.

The invention has the following beneficial effects:

1. the invention establishes a stable manufacturing method of hyaluronic acid fragment B-HA with the average molecular weight of 35 +/-8 KDa, and discovers a manufacturing principle and a new action mechanism of the hyaluronic acid fragment B-HA.

2. The invention finds the biological property that the hyaluronic acid fragment rapidly enters lymph nodes and spleen of lymph organs after subcutaneous and intravenous injection, and develops new application based on the property.

3. The invention discovers that the hyaluronic acid fragments promote the removal of human mononuclear cells (mainly human lymphocytes and a small number of human monocytes), and suggests that the hyaluronic acid fragments carry moisture together with the human mononuclear cells from inflammatory tissues into lymphatic return and immunoregulation of the lymphocytes.

4. The invention discovers that the hyaluronic acid fragment obviously inhibits the removal of neutrophils under the condition of higher concentration, and suggests that the hyaluronic acid fragment has anti-inflammatory effect when being locally used.

5. The invention discovers that the hyaluronic acid fragment has good safety in intravenous injection; the molecular weights of the electrophoresis gel of the hyaluronic acid fragments which are the final products of the high molecular hyaluronic acid raw materials are both about 35kDa after the full or slight excess enzymolysis of the hyaluronic acid fragments of the high molecular hyaluronic acid raw materials in human hyaluronidase PH20 and bovine-extracted hyaluronidase PH 2010 minutes, 20 minutes, 40 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours; of these, fragments of 35kDa hyaluronic acid of substantially the same molecular weight were produced at 2,3, 4,5 and 6 hours, and were able to be filtered using 0.22 μm (220nm) pore size filters to ensure good tissue permeability, supporting good tissue permeability.

6. The invention discloses a method for preparing hyaluronic acid fragments by directly mixing commercial hyaluronidase PH20 injection extracted from bovine testes with commercial polymer hyaluronic acid injection, and application of the method in preparing medicaments for promoting lymphoedema and cell reflux and local anti-inflammatory, wherein the hyaluronidase PH20 injection extracted from the bovine testes can be clinically used for preparing the hyaluronic acid fragments by human allergy experimental skin test.

7. The invention discloses a method for preparing hyaluronic acid fragments with different molecular weights by using hyaluronidase with different molecular weights, which is prepared by recombining and extracting various species of full-length, partial and fused hyaluronidase with different molecular weights, and is characterized in that the biological effects of the hyaluronic acid fragments with different molecular weights are different.

Drawings

The foregoing is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and the detailed description.

FIG. 1 shows the results of SDS-PAGE electrophoresis detection of recombinant human hyaluronidase PH20 obtained after 4-step purification of recombinant human hyaluronidase PH20 produced in CHO cells using a GC-rich animal cell expression vector by a QFF chromatography column, a Phenyl HP hydrophobic chromatography column, a CHT I type ceramic hydroxyapatite chromatography column and an SPHP cation exchange chromatography column.

FIG. 2 shows gel electrophoresis results of hyaluronic acid fragment final products prepared by enzymatically cleaving a raw material of a high molecular weight hyaluronic acid using a sufficient amount or a slight excess of recombinant human hyaluronidase PH 2010 min (Lane-1), 20 min (Lane-2), 40 min (Lane-3), 1 hr (Lane-4), 2 hr (Lane-5), 3 hr (Lane-6), 4 hr (Lane-7), 5 hr (Lane-8), and 6 hr (Lane-9). Lane-10 and Lane-11 are 24kDa and 35kDa hyaluronic acid fragment standards, respectively.

FIG. 3 shows gel electrophoresis results of hyaluronic acid fragment final products prepared by enzymatically cleaving a raw material of a high molecular weight hyaluronic acid using hyaluronidase extracted from cattle in a sufficient amount or a slight excess amount, PH 2010 minutes (Lane-1), 20 minutes (Lane-2), 40 minutes (Lane-3), 1 hour (Lane-4), 2 hours (Lane-5), 3 hours (Lane-6), 4 hours (Lane-7), 5 hours (Lane-8), and 6 hours (Lane-9). Lane-10 and Lane-11 are 24kDa and 35kDa hyaluronic acid fragment standards, respectively.

FIG. 4 shows gel electrophoresis results of hyaluronic acid fragment final products prepared by enzymatically cleaving a polymeric hyaluronic acid raw material using a non-PH 20 recombinant hirulog hyaluronidase in a sufficient amount or in a slight excess amount for 10 minutes (Lane-1), 20 minutes (Lane-2), 40 minutes (Lane-3), 1 hour (Lane-4), 2 hours (Lane-5), 3 hours (Lane-6), 4 hours (Lane-7), 5 hours (Lane-8), and 6 hours (Lane-9). Lane-10 and Lane-11 are 24kDa and 35kDa hyaluronic acid fragment standards, respectively.

FIG. 5 shows the results of gel electrophoresis molecular weight measurement of hyaluronic acid fragment B-HA products of 6 different production lots, and the molecular weights of the B-HA products of 6 different lots marked in the figure are the results of GPC-RI-MALLS molecular weight measurement (see Table 3).

FIG. 6 is a graph of the primary distribution of 99mTc-B-HA after 5 minutes intravenous injection; wherein the 5 minutes later, the antibody is mainly distributed in spleen, which indicates that 99mTc-B-HA HAs affinity to lymph tissue; 99mTc-B-HA was also predominantly distributed in the liver after 5 minutes of intravenous injection; 99mTc-B-HA also distributed in the bladder 5 minutes after intravenous injection.

FIG. 7 is a planar image taken 5 minutes after subcutaneous injection of mice; wherein, each C57BL/6J mouse is injected with 20-25 mu Ci of 125I-B-HA subcutaneously at the end of the lower limb, planar images are collected by PET-CT of the small animal every 5, 10, 30 and 140 minutes, and the result of 5 minutes collection shows that 125I-B-HA rapidly enters lymph node and lymph organ spleen within 5 minutes by subcutaneous injection at the end of the lower limb of the C57BL/6J mouse.

FIG. 8 is a bar graph comparing different substances to facilitate removal of mononuclear cells; wherein the removal of freshly extracted mononuclear cells (predominantly lymphocytes and a small number of macrophage precursor mononuclear cells) is facilitated by higher concentrations (300 μ g/mL) of 35kDa hyaluronic acid B-HA and 1600kDa hyaluronic acid HA; the 35kDa hyaluronic acid fragment B-HA mixed with 1600kDa hyaluronic acid HA further facilitated the removal of freshly extracted mononuclear cells. Anti-human CD44antibody, endotoxin LPS and chemokine fMLP also all facilitated the removal of freshly extracted mononuclear cells. Two sets compare p <0.05 (n-4); two sets compare p <0.01(n ═ 4); two sets compare p <0.001(n ═ 4).

FIG. 9 is a bar graph comparing different substances to facilitate removal of mononuclear cells; among these, endotoxin LPS facilitates the removal of freshly extracted mononuclear cells from humans. Higher concentrations (300. mu.g/mL) of hyaluronic acid HA further facilitated endotoxin LPS (1ng/mL) induced removal of freshly extracted human mononuclear cells. Higher concentrations (300. mu.g/mL) of the hyaluronic acid fragment B-HA did not significantly affect endotoxin LPS (1ng/mL) induced human freshly extracted monocyte removal. However, the higher concentration (300. mu.g/mL) of hyaluronic acid fragment B-HA significantly inhibited the endotoxin LPS mixing with the higher concentration (300. mu.g/mL) of hyaluronic acid HA facilitated the removal of human freshly extracted mononuclear cells. Two sets compare p <0.05(n ═ 4), two sets compare p <0.01(n ═ 4), and two sets ns compare p >0.05(n ═ 4).

FIG. 10 is a bar graph comparing the inhibition of neutrophil migration by different substances; of these, the higher concentration (300. mu.g/mL) of 35kDa hyaluronic acid fragment B-HA, 1600kDa hyaluronic acid HA and B-HA mixed HA all inhibited the removal of freshly extracted neutrophils, suggesting that HA and B-HA have the same affinity and biological effect on the hyaluronic acid receptors CD44 and Siglec-9 of neutrophils. The anti-CD44antibody, endotoxin LPS and chemokine fMLP all promoted the removal of freshly extracted neutrophils to varying degrees. Two sets compare p <0.05(n ═ 4), two sets compare p <0.01(n ═ 4).

FIG. 11 is a histogram comparing the inhibition of neutrophil migration by different substances; wherein the higher concentration (300. mu.g/mL) of 35kDa hyaluronic acid fragment B-HA, 1600kDa hyaluronic acid HA and the mixed HA of B-HA all significantly inhibited endotoxin LPS (1ng/mL) induced removal of human freshly extracted neutrophils. Endotoxin LPS and chemokine fMLP facilitate the removal of freshly extracted human neutrophils. Two sets compare p <0.001(n ═ 4).

Detailed Description

The present invention is illustrated by the following specific examples, which are presented herein for the purpose of illustration and explanation and are not intended to be limiting.

Through a large number of literature studies by the applicant, the interaction between hyaluronic acid and hyaluronic acid fragments and various hyaluronic acid receptors of human bodies, including LYVE-1, CD44, RHAMM, Siglec-9, HARE, TLR2, CEMIP and TMEM2, is involved in the regulation of various functions in vivo, such as lymphocyte trafficking, secretion of leukocyte inflammatory factors, tumor immune regulation, cardiovascular and cerebrovascular tissue renewal and the like. It is worth to suggest that the clinical studies of bioactive hyaluronic acid fragments B-HA with an average molecular weight of around 35kDa are completely different between injection (contacting various tissues and organs) and topical application to the skin mucosa (limited penetration contacting the skin mucosa from the outside) with good tissue penetration. The clinical effect of the bioactive hyaluronic acid fragment B-HA with good tissue permeability and average molecular weight of about 35kDa on human bodies after injection is the comprehensive result of the combination of different cells and different tissues and organs with various receptors.

Therefore, a hyaluronic acid fragment B-HA injection with good tissue permeability is developed, and the important pharmaceutical and clinical significance of the action mechanism and the comprehensive action of the hyaluronic acid fragment and a plurality of hyaluronic acid receptors in different cells, tissues and organs after the hyaluronic acid fragment is absorbed by a human body is further clarified.

Example 1

The purpose is as follows: the preparation method of hyaluronic acid fragments with different low molecular weights by using different hyaluronidases to sufficiently or slightly excessively and sufficiently enzymolyze a high molecular weight hyaluronic acid raw material is researched.

The method comprises the following steps:

1. production of recombinant human hyaluronidase PH20

Based on the method described in reference [49], a cDNA of artificially synthesized recombinant human hyaluronidase PH20 was inserted into a GC-rich pMH3 vector to construct a pMH3-PH20 expression vector; then transferring the pMH3-PH20 expression vector into a CHO-S cell line, screening the CHO-S cell line with high expression PH20, and carrying out amplification and large-scale culture in a torrent type animal cell reactor; filtering the harvest liquid containing PH20 with 0.22 μm filter membrane, purifying with QFF chromatographic column, Phenyl HP hydrophobic chromatographic column, CHT I type ceramic hydroxyapatite chromatographic column and SP HP cation exchange chromatographic column for 4 steps, and filtering with 0.22 μm filter membrane to obtain sterile saccharified recombinant human hyaluronidase PH20[50,51] with purity of more than 98.5%.

2. Production of hyaluronic acid fragment B-HA

Human hyaluronidase PH20 is a sperm acrosome hyaluronidase produced in male testis [43], and also produced in female breast [52 ]. The human colostrum contains a hyaluronic acid fragment B-HA (53-57) of 35kDa with certain anti-inflammatory activity, which is prepared by cutting high molecular hyaluronic acid with PH20, and the hyaluronic acid fragment B-HA is produced by using an enzymolysis reactor with a working volume of 25 liters and cleaned and sterilized. Firstly, preparing an injection-grade hyaluronic acid raw material with the average molecular weight of 1600kDa, adding the hyaluronic acid raw material into water for injection once or for multiple times, then sequentially adding sodium chloride (with the final concentration of 80-90mmol/L), magnesium ions (with the final concentration of 1mmol/L) and recombinant human hyaluronidase (with the final concentration of 15000 units/g), fully mixing, reacting at 37 ℃ for 30 minutes, 1 hour, 2 hours, 3 hours and 4 hours respectively, sampling and determining the molecular weight of an enzymolysis product, and taking the hyaluronic acid fragment with the average molecular weight of 35kDa as the optimal enzymolysis time. And then sodium chloride (35-45mmol/L) is added to adjust the osmotic pressure to 280-300 milliosmol/L (mOsm/L), the residual recombinant human hyaluronidase PH20 is heated for 15 minutes at 85 ℃ for heat inactivation, 2 percent (20mg/mL) hyaluronic acid fragment B-HA solution is obtained by filtering through a 0.22 mu m filter membrane, and the sample is sent for endotoxin content, residual protein, residual nucleic acid and glucuronic acid content detection and bacterial culture.

3. Molecular weight determination of hyaluronic acid fragment B-HA

(1) Agarose gel electrophoresis method of determination

Dissolving 0.3g of agarose in 30mL of TBE solution, heating and boiling for more than three times in a microwave oven, slightly cooling, pouring into a gel making plate inserted with a gel making comb, and cooling and solidifying the gel. And adding 45 mu L of ultrapure water into 15 mu L of standard substance to prepare a standard substance solution, adding 15 mu L of ultrapure water into 5 mu L of sample to be detected to prepare a sample solution, wherein the final concentrations of the standard substance and the sample are both 5mg/mL, and respectively and uniformly mixing the standard substance and the sample with the sample buffer according to a ratio of 4: 1. The prepared gel plate is placed in an electrophoresis tank containing 1 XTBE, 20 microliter of standard substance and sample are added into each gel hole in sequence, the voltage of an electrophoresis apparatus is adjusted to 80v, and electrophoresis is carried out for 20 minutes.

(2) Measurement by GPC-RI-MALLS method

Analyzing the molecular weight of a sample by adopting a Gel Permeation Chromatography (GPC) -differential detection (RI) eighteen-angle laser scattering instrument (MALLS) combined on-line detection method, (1) chromatographic conditions, namely an instrument, namely a high performance liquid chromatograph (matched with an RI detector); shodex SB-804HQ gel column (phi 8mm x 300 mm); the mobile phase is 0.02 percent sodium azide aqueous solution; the flow rate is 1 mL/min; the sample injection amount is 100 mu L; the temperature is 40 ℃; the detector is a differential detector and an eighteen-angle laser scattering detector. (2) And (3) measuring the relative molecular mass of the sample, namely weighing a proper amount of sample, dissolving the sample by using a mobile phase to prepare a 1mg/mL polysaccharide sample solution, filtering the solution by using a 0.22 mu m filter, injecting the sample, and performing high performance liquid analysis. (3) The molecular mass distribution detection adopts an RID and a laser detector to respectively record the mass concentration of a test sample and the light scattering intensity of the test sample at different angles, the value of the refractive index increment (dn/dc) is 0.138, and a molecular mass distribution diagram of the test sample is obtained through a data processing software ASTRA.

4. Hyaluronic acid raw material

Hyaluronic acid raw material (Wash-Fridad Biotech Co., Ltd.) having an average molecular weight of 300kDa (medium molecule), 1600kDa (high molecule) and sodium hyaluronate or hyaluronic acid injection (trade name of Schonfert) having an average molecular weight of 1600kDa (Bausch & Lomb.

5. Hyaluronidase

Recombinant human hyaluronidase PH20 (Shaoxing Hui technologies, Inc.), recombinant human soluble hyaluronidase PH20-IgG2Fc (Hangzhou Anrapu biopharmaceutical research, Inc.), extracted bovine testis hyaluronidase PH20(1500 u/ramus, H31022111, Shanghai first chemical pharmaceutical factory), and recombinant leech hyaluronidase (Jiangnan university). The above hyaluronidase activity assay method is different, and the present invention is based on the activity marker provided by the supplier. Recombinant hirulog hyaluronidase is described in detail in the references Yuan Panhong, Kang Zhen, Jin Pen, Liu Long, Zhu Guiche, Chen Jian, Preparation of small hydrolona by enzymic hydrolysics in vitro (Chinese), Journal of Food Science and Technology,2016,34(2): 51-55; peng Jin, Zhen Kang, Na Zhang, Guoheng Du, and Jian Chen, High-yield novel horizontal uronidase to expect the preparation of specific hyaluronic oligomers, Scientific Reports, DOI:10.1038/srep04471 (2014).

6. Method for preparing low molecular hyaluronic acid fragments by sufficient or slight excessive enzymolysis

The preparation method comprises the steps of firstly determining the using amount of recombinant human hyaluronidase PH20 (hereinafter referred to as PH20), recombinant human hyaluronidase PH20 and IgG2Fc fusion protein (hereinafter referred to as PH20-IgG2Fc), extracting sufficient or slightly excessive enzyme for sufficient enzymolysis of bovine testis hyaluronidase PH20 and recombinant leech hyaluronidase by adopting a pre-experiment, and then reproducing and manufacturing. Hyaluronidase is defined as being sufficiently digested in either a sufficient amount or a slight excess of hyaluronidase as: (1) the molecular weight of the hyaluronic acid fragments prepared by sufficiently or slightly excessively performing enzymolysis on the neutralized high molecular weight hyaluronic acid raw material for 10-20 minutes is basically consistent with that of the hyaluronic acid fragments prepared by sufficiently or slightly excessively performing enzymolysis on the neutralized high molecular weight hyaluronic acid raw material for 1-6 hours (the coefficient of variation CV is less than 15%); (2) the low molecular hyaluronic acid fragment product of which more than 99 percent of high or medium molecular hyaluronic acid raw material is sufficiently or slightly excessively and sufficiently enzymolyzed is completely and smoothly filtered by a 022um filter membrane; (3) hyaluronidase activity was completely inactivated at 80 degrees for 45 minutes with little or no residue (< 15%) after sufficient or mild excess enzymatic reaction. Various time points for enzymatic production of hyaluronic acid include: enzymolysis is carried out for 10 minutes, 20 minutes, 40 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours. The recombinant human hyaluronidase pH20 and the extracted bovine testicular hyaluronidase pH20 were used for enzymatic hydrolysis at pH 7.4. Recombinant leech hyaluronidase was used pH 6.5.

As a result:

1. the SDS-PAGE results of the purified recombinant human hyaluronidase PH20 produced using the GC-rich animal cell expression vector are shown in FIG. 1. The purity of the recombinant Human PH20 is more than 98.5% by a size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) detection method, and the specific activity is 56802 +/-508 IU/mg (n is 3) by a Human HAase Elisa kit detection method.

2. Human hyaluronidase PH20(15000 units) (fig. 2), bovine-derived hyaluronidase PH20(20000 units) (fig. 3) and recombinant hirulog hyaluronidase (150000 units) (fig. 4) were mixed in a solution containing 140mmol/L NaCl and 1mmol/L MgCl and incubated at 37 ℃ for 10 minutes, 20 minutes, 40 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours, respectively, suggesting that the optimal enzymatic hydrolysis times for human hyaluronidase PH20 and bovine-derived hyaluronidase PH20 were 2,3, 4 and 5 hours, and the molecular weights of the enzymatic hydrolysis end products were 35kDa (35 ± 8 kDa). Note: dosage units using a sufficient or slight excess of human hyaluronidase PH20, bovine extracted hyaluronidase PH20(20000 units) and recombinant leech hyaluronidase were derived from preliminary experiments.

Table 1 hyaluronic acid fragment solutions prepared by enzymatically cleaving a polymeric hyaluronic acid raw material using 0.22 μm filter membrane filtration of human hyaluronidase PH 2010 minutes, 20 minutes, 40 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours, tissue permeability of each group of hyaluronic acid fragment solutions and the ability to filter-sterilize using 0.22 μm filter membrane were examined. The results of the study in table 1 show that the average molecular weight 35kDa hyaluronic acid fragment solutions prepared by enzymatic hydrolysis of the bulk or slight excess of the polymeric hyaluronic acid starting material with human hyaluronidase PH 2010 minutes, 20 minutes, 40 minutes and 1 hour were not able to complete a 0.22 μmembrane filtration without significant resistance, suggesting that a portion of the hyaluronic acid fragments with larger molecular weights were not able to pass through the interstitial spaces of the 220nm pore size. The results of the study in table 1 also show that the average molecular weight hyaluronic acid fragment solutions prepared by sufficiently digesting the polymeric hyaluronic acid starting material with sufficient or slight excess of human hyaluronidase PH20 for 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours were all able to complete 0.22 μm membrane filtration without significant resistance, suggesting that there were no hyaluronic acid fragments in the hyaluronic acid fragment solution having a larger molecular weight that could not pass through the interstitial spaces of the 220nm pore size. The above results suggest that the 0.22 μm membrane filtration of the hyaluronic acid fragment solution is a method for determining whether or not the average molecular weight hyaluronic acid fragments produced by enzymatic hydrolysis of a polymeric hyaluronic acid raw material with human hyaluronidase PH20 are sufficient, and also a method for determining whether or not the hyaluronic acid fragment solution can be sterilized by filtration with a 0.22 μm membrane.

Table 2 shows the molecular steric characteristics, molecular weights and amino acid numbers of the hyaluronic acid fragments produced by sufficiently or slightly excessively hydrolyzing the polymeric hyaluronic acid material with full-length, partial and fused recombinant and extracted hyaluronidase of different species. The results in table 2 suggest that hyaluronidase enzymatic cleavage of polymeric hyaluronic acid raw material using full length, partial and fused recombinant and extracted hyaluronidase from a variety of different species produces fragments of hyaluronic acid with varying molecular stereo-structures and molecular weights. The receptor binding capacity and biological effect of the hyaluronic acid fragments prepared by enzymolysis and cutting with different stereo structures and molecular weights are different. The molecular stereo structure and the number of amino acids of the human hyaluronidase PH20 are similar, and the molecular weight of the hyaluronic acid fragments prepared by enzymolysis cutting of the high molecular hyaluronic acid raw material is the same. The results in table 2 also show that the molecular weight of the hyaluronic acid fragments produced by enzymatically cleaving the polymeric hyaluronic acid starting material with hirudin hyaluronidase is significantly different from the molecular weight of the hyaluronic acid fragments produced by enzymatically cleaving the polymeric hyaluronic acid starting material with human hyaluronidase PH20 (fig. 2,3, 4). The results in table 2 further show that the molecular weight of the hyaluronic acid fragments prepared by enzymatically cleaving the polymeric hyaluronic acid starting material with the recombinant human soluble hyaluronidase PH20-IgG2Fc is also significantly different from the molecular weight of the hyaluronic acid fragments prepared by enzymatically cleaving the polymeric hyaluronic acid starting material with the human hyaluronidase PH 20.

TABLE 2 results of molecular stereostructural characteristics, molecular weights and amino acid numbers of hyaluronic acid fragments produced by enzymatic hydrolysis of polymeric hyaluronic acid starting material with full or partial fusion of full, partial and fused recombinant and extracted hyaluronidases of different species

3. Molecular weight determination of hyaluronic acid fragment B-HA

(1) Agarose gel electrophoresis assay

The gel electrophoresis results of the hyaluronic acid fragment B-HA products produced in 6 different batches are shown in FIG. 3, wherein the molecular weights of the products marked in the figure are the results of GPC-RI-MALLS measurement in Table 3, the gel electrophoresis results are basically consistent with the results of GPC-RI-MALLS measurement, and the average molecular weight is distributed at about 35 kDa.

(2) GPC-RI-MALLS assay

The average molecular weight of the hyaluronic acid fragments B-HA products of 6 different production batches was determined by GPC-RI-MALLS (table 3), and the results showed that the average molecular weight of 6 samples was 35 ± 8kDa, with an interpass variation CV of 22% (n ═ 6), consistent with the molecular weight of hyaluronic acid fragments extracted from human colostrum reported in the literature [51-55 ].

TABLE 3 average molecular weight of hyaluronic acid fragment B-HA products of 6 different production batches determined by GPC-RI-MALLS

Molecular weight distribution of hyaluronic acid fragment B-HA products of 6 different production batches was determined using GPC-RI-MALLS (Table 4, Table 5), and the results showed that 92.75 + -2.42% of B-HA was distributed between 10-70kDa and 96.92 + -2.34% of B-HA was distributed between 10-100kDa, with a relatively small distribution range.

TABLE 4 GPC-RI-MALLS results of 10-70kDa molecular weight distribution of 6 different batches of hyaluronic acid fragment B-HA products

TABLE 5 GPC-RI-MALLS results of 10-100kDa molecular weight distribution of 6 different batches of hyaluronic acid fragment B-HA products

Discussion:

the results of the invention show that the molecular weight of the hyaluronic acid fragments prepared by sufficiently or slightly excessively and sufficiently hydrolyzing the raw material of the high molecular weight hyaluronic acid with different hyaluronidases (recombinant human hyaluronidase PH20, recombinant human soluble hyaluronidase PH20-IgG2Fc, extracted bovine hyaluronidase PH20 and recombinant leech hyaluronidase) are obviously different (table 2, figures 2,3 and 4), and the molecular weight and the function of the hyaluronic acid fragments prepared by sufficiently or slightly excessively and sufficiently hydrolyzing the raw material of the high molecular weight hyaluronic acid with different hyaluronidases are different. The above findings further suggest that the molecular weight and biological effect of the hyaluronic acid fragments produced by enzymatic cleavage of various hyaluronidases (including molecular steric structure, molecular weight, number of amino acids and variety are not completely the same as those of PH20 or Hyal-5 or SPAM1, Hyal-1, Hyal-2, Hyal-3 and Hyal-4) in human body are not completely the same.

The results of the studies of fig. 2,3 and 4 also show for the first time that the length of time for which the hyaluronidase sufficiently or slightly excessively sufficiently attacks the raw material of the high molecular weight hyaluronic acid is not closely related to the molecular weight of the produced hyaluronic acid fragment, and that the molecular weight of the hyaluronic acid fragment produced by sufficiently attacking the raw material of the high molecular weight hyaluronic acid for a short time of 10 to 20 minutes is substantially the same as the molecular weight of the hyaluronic acid fragment produced by sufficiently attacking the raw material of the high molecular weight hyaluronic acid for a long time of 1 to 6 hours (gel electrophoresis measurement result). This finding indicates that sufficient enzymatic hydrolysis of a high molecular weight hyaluronic acid starting material using recombinant human hyaluronidase PH20 in sufficient quantities or in slight excess can stably produce hyaluronic acid fragments with an average molecular weight of 35kDa or close (fig. 5, tables 3, 4, 5).

The results of the study in table 1 show for the first time that the average molecular weight 35kDa hyaluronic acid fragment solutions prepared by enzymatic hydrolysis of a polymeric hyaluronic acid starting material with sufficient or slight excess of human hyaluronidase PH 2010 minutes, 20 minutes, 40 minutes and 1 hour do not completely complete 0.22 μm membrane filtration without significant resistance, suggesting that the hyaluronic acid fragment solutions prepared have a larger molecular weight and cannot pass the hyaluronic acid fragments in the interstitial space of 220nm pore size tissue. The results of the study in table 1 also show for the first time that the solutions of hyaluronic acid fragments with average molecular weight of 35kDa produced by enzymolysis of the polymeric hyaluronic acid starting material with sufficient or slight excess for 202 hours, 3 hours, 4 hours, 5 hours and 6 hours are all capable of complete 0.22 μm membrane filtration without significant resistance, suggesting that the produced solutions of hyaluronic acid fragments do not have hyaluronic acid fragments with larger molecular weight that cannot pass through the interstitial space of tissue with pore size of 220 nm. The above results suggest that the 0.22 μm membrane filtration is a method for determining whether the average molecular weight 35kDa hyaluronic acid fragment produced by hydrolyzing the polymeric hyaluronic acid raw material with a sufficient amount or a slight excess of human hyaluronidase PH20 is sufficiently complete, and also a method for determining whether the hyaluronic acid fragment can be sterilized by filtration with a 0.22 μm membrane.

Among all known hyaluronidases, hyaluronidase PH20 is the only hyaluronidase that reacts at neutral PH and is structurally specific with two binding sites, namely the N-terminal hyaluronic acid binding site and the C-terminal egg zona pellucida binding site. In other words, PH20 is a binding protein for both hyaluronic acid and zona pellucida. The findings in table 2 suggest that hyaluronidase enzymatic cleavage of polymeric hyaluronic acid raw materials using full-length, partial and fused recombinant and extracted hyaluronidases from a variety of different species produces hyaluronic acid fragments with molecular stereostructures, molecular weights of varying sizes and biological effects that are not exactly the same. The molecular stereo structure, molecular weight and amino acid number of the human and bovine hyaluronidase PH20 are similar, and the receptor binding and biological effects of the hyaluronic acid fragments prepared by enzymolysis and cutting of a high molecular hyaluronic acid raw material are also possible. For example, the molecular weight of hyaluronic acid fragments produced by enzymatic digestion of hyaluronidase from different sources in sufficient amounts or in slight excess is also related to biological activity and receptor binding activity (Cyphert JM, Trempus CS, Garntziosis S: Size matrices: molecular weight specificity of hyaluronic acids in Cell biology (Review Article). Int J Cell Biol 2015,2015: 1-8.).

In summary, the recipe for sufficient or slight excess of sufficient enzymatic hydrolysis of recombinant human hyaluronidase PH20 and extracted bovine hyaluronidase PH20 is defined as: (1) the molecular weight of the hyaluronic acid fragments prepared by sufficiently performing enzymolysis neutralization on the high molecular hyaluronic acid raw material for 10-20 minutes in a short time and the molecular weight of the hyaluronic acid fragments prepared by sufficiently performing enzymolysis neutralization on the high molecular hyaluronic acid raw material for 1-6 hours in a long time are basically consistent (the coefficient of variation CV is less than 15%); (2) the high molecular hyaluronic acid raw material with 99 percent of neutralization is sufficiently enzymolyzed into a low molecular hyaluronic acid fragment product which can be effectively filtered by a 0.22um filter membrane by sufficient amount or slight excess; (3) hyaluronidase activity is substantially free or very low (< 15%) after sufficient enzymatic hydrolysis with sufficient or mild excess, and can be inactivated at 80 degrees for 45 minutes.

Literature studies have shown that fragments of hyaluronic acid extracted from human colostrum have an average molecular weight of 35kDa and are most probably produced by the sperm acrosome hyaluronidase PH20 of male gonadal testes. The invention shows that the hyaluronic acid fragment with the average molecular weight of 35kDa and prepared from female breast can be stably obtained by sufficiently or slightly excessively hydrolyzing hyaluronic acid raw materials with the molecular weight of 1600kDa by sperm acrosome hyaluronidase PH20 of recombined human male gonadal testis and hyaluronidase PH20 extracted from bovine testis (figures 2 and 3). The results of the invention show that sufficient or slight excess of PH20 is used for sufficiently digesting a hyaluronic acid raw material with the molecular weight of 1600kDa in a certain time range, so that hyaluronic acid fragments with the average molecular weight of 35kDa with small up-down variation are obtained (figure 5; Table 3). Therefore, the invention establishes a method for preparing the hyaluronic acid fragment B-HA with the average molecular weight of 35kDa by cutting the hyaluronic acid raw material with the average molecular weight of 1600kDa by using the stable recombinant human hyaluronidase PH20, and the preparation principle is clear. In other words, the present application reports for the first time that the average molecular weight of the enzymatic hydrolysate produced by enzymatically cleaving a starting hyaluronic acid material using human and bovine hyaluronidase PH20 in sufficient quantities or in slight excess is 35kDa with little variation.

And (4) conclusion: 1. the molecular weights of the electrophoresis gel of the hyaluronic acid fragments of the final product of the high molecular hyaluronic acid raw material are both 35kDa (35 +/-8 KDa) after the full or slight excess enzymolysis of the hyaluronic acid fragments of the high molecular hyaluronic acid raw material in the steps of human hyaluronidase PH20 and bovine-extracted hyaluronidase PH 2010 minutes, 20 minutes, 40 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours; 2. using human hyaluronidase PH20 to perform enzymolysis and cutting of macromolecule hyaluronic acid raw material with average molecular weight of 1600kDa for 2 hours, 3 hours, 4 hours and 5 hours, stably manufacturing 35kDa hyaluronic acid fragments with basically the same molecular weight, and showing that the hyaluronic acid fragments can be filtered by a 0.22 mu filter membrane; 3. the stably manufactured 35kDa hyaluronic acid fragment is filtered by using a 0.22 mu filter membrane to verify and ensure the tissue permeability; 4. the molecular weight of the prepared high molecular hyaluronic acid raw material is different by using the hyaluronidase which is prepared by full-length, partial and fused recombination and extraction of various different species, and the biological effects of the hyaluronic acid raw material can also be different.

Example 2

The purpose is as follows: the rapid absorption route in vivo by subcutaneous and intravenous injection of 125-I and 99 mTc-labeled mice was investigated for hyaluronic acid fragments (hereinafter referred to as B-HA or HA35) having an average molecular weight of 35kDa, and further investigated for their novel therapeutic action and novel mechanism of action using biomolecular imaging techniques and human freshly extracted human neutrophils and monocytes (mainly lymphocytes and a small number of monocytes).

Experimental reagents, equipment, human and animal blood cell samples and experimental animals:

1. experimental reagent: injection grade hyaluronic acid raw material with an average molecular weight of 1600kDa, ordinary hyaluronic acid raw material with an average molecular weight of 300kDa and hyaluronic acid fragment with an average molecular weight of 24kDa were purchased from Huanxifurada biomedical Co., Ltd, hyaluronic acid fragment with an average molecular weight of 60kDa was purchased from Liyangyang Biotech Co., Ltd, Fetal Bovine Serum (FBS) was purchased from Hangzhou Biotech Co., Ltd, Penicillin-Streptomycin Solution was purchased from Hyclone Co., USA, anti-CD44antibody, rabbit IgG was purchased from Abcam Co., Ltd (UK), Cy5.5 fluorescent dye, Lipopolysaccharide (LPS), Agarose (Agarose), phorbol ester (PMA), chemokine (fMLP) was purchased from Beijing Sorbao Tech Co., Ltd, RPMI buffer-1640 medium, PBS buffer was purchased from PBS-Aldrich Co., USA, human neutral granulocyte isolation was purchased from Tianjin ocean biological products Ltd, TNF-alpha Elisa Kit was purchased from R & D Systems (USA), Human HAase Elisa Kit was purchased from Shanghai enzyme-linked Biotechnology Ltd, and SDS-PAGE gel preparation Kit was purchased from Hakka century Bio Inc.

2. An experimental instrument: enzymatic hydrolysis reactor (hangzhou anpu biotechnology limited), osmolarity measuring instrument (tianhe medical instruments limited), torrent animal cell reactor was provided by shaohui aloe biotechnology limited, recombinant human hyaluronidase PH20 was purified using QFF chromatography column, Phenyl HP hydrophobic chromatography column, CHT I type ceramic hydroxyapatite chromatography column, SP HP cation exchange chromatography column was provided by the institute of biotechnology of military medical sciences, flow cytometer (BD Bioscience, usa) was provided by the central laboratory of Qingdao agricultural university, inverted microscope was provided by south kyo Jiangnan Yongxin optical limited, eighteen degree laser scattering instrument was provided by Wyatt Technology company (usa), differential detector was provided by Wyatt Technology company (usa), black background 2016 type microscope was provided by haar Bingkang Black background optical instrument limited.

3. Human blood cell samples: the healthy volunteers were 12 persons, aged 24 ± 4 years, and were approved by vinpocetine and ethics committee of medical care in surgical hospitals and with consent of the same.

4. Animal red blood cell samples: venous blood from beagle dogs, BALB/c mice, inner Mongolia goats, and inner Mongolia cattle was provided by the animal Hospital, Qingdao university of agriculture.

5. Experimental animals: experimental C57BL/6J mice, beagle dogs, were provided by Arizona State University and Qingdao University animal Hospital, respectively.

The method comprises the following steps:

1. 99mTc and 125-I labeling of hyaluronic acid fragment B-HA and study of tissue distribution and molecular imaging

(1) 99mTc labeling and tissue distribution studies of hyaluronic acid fragment B-HA: tc (V) is reduced to [ TcOCl4] -, using SnCl2, and binds to the carboxyl group of B-HA to form a 99 mTc-labeled B-HA stabilizing compound (hereinafter 99 mTc-B-HA). After SEC-HPLC purification analysis, the purity of the injection 99mTc-B-HA with the purity of more than 98 percent is obtained. Intravenous injection of purified 99mTc-B-HA was administered via a pre-catheter, healthy C57BL/6J mice (The Jackson Laboratory, USA) aged 6-8 weeks. A novel iQID gamma camera developed by the university of Arizona is used for dynamically acquiring 99mTc-B-HA whole body tissue distribution images for three hours. After the iQID image collection was completed, mouse blood and tissue organ samples were collected for radioactivity content determination. The 99mTc-B-HA distribution of mouse tissue organs was expressed as a percentage of the total injected dose (% ID/g).

(2) 125-I labeling and molecular imaging studies of hyaluronic acid fragment B-HA: 125I and 35kDa hyaluronic acid fragment B-HA are covalently connected by using an Iodogen method, and the manufactured 125I-B-HA is purified by a molecular sieve column and eluted by PBS for later use. Each C57BL/6J mouse was injected subcutaneously at the end of the lower extremities with 20-25. mu. Ci of 125I-B-HA, and planar images were acquired by small animal PET-CT every 5, 10, 30, 140 minutes, with 5 minute gamma radioactive counts each.

2. Effect of higher concentrations of hyaluronic acid fragments B-HA on human neutrophil and monocyte removal in lymph fluid

(1) Effect of higher concentration of hyaluronic acid fragments B-HA on the removal of freshly extracted mononuclear cells (mainly lymphocytes and a small number of macrophage precursor mononuclear cells) by humans: freshly extracted human mononuclear cells were used and resuspended in 1 × RPMI-1640 medium, adjusting the cell density to 3 × 108 cells/mL. Placing 0.8% agarose sterilized solution in 37 deg.C water bath, mixing equal amount of agarose solution with 2 × RPMI-1640 culture medium containing 20% FBS and 1% streptomycin, mixing equal amount of above mixed solution with mononuclear cell suspension, and placing in 37 deg.C water bath. A96-well plate precooled at-20 ℃ is taken, 2 mu L of the mononuclear cell agarose mixed solution is added into each well, and a glue drop with the diameter of about 2mm is required to be formed at the center of the bottom of each well. The gel-drop-spread 96-well plate was placed at 4 ℃ for 15 minutes. RPMI-1640 medium containing 10% FBS, 1. mu.g/mL Anti-CD44Anti body + 10% FBS, 300. mu.g/mL B-HA + 10% FBS, 300. mu.g/mL B-HA + 300. mu.g/mL HA + 10% FBS, 1ng/mL LPS + 10% FBS, 1nmol/L fMLP + 10% FBS was prepared, and after the agarose gel drops in the wells had solidified, 100. mu.L of the above reagents were added to each well separately, 4 replicates in each group. And culturing the 96-well plate in a 37 ℃ constant temperature incubator for 3 hours, taking out, observing the migration result under an inverted microscope, photographing and recording, and calculating the cell migration area by using Image processing software Image j. Venous blood from 3 different volunteers per collection excluded individual differences and ensured that the experiment was reproducible.

(2) Effect of higher concentration of hyaluronic acid fragment B-HA on LPS-induced removal of human monocytes (mainly lymphocytes and a small amount of macrophage precursor monocytes) freshly extracted human monocytes were used and resuspended in 1 × RPMI-1640 medium, adjusting the cell density to 3 × 108 cells/mL. Placing 0.8% agarose sterilized solution in 37 deg.C water bath, mixing equal amount of agarose solution with 2 × RPMI-1640 culture medium containing 20% FBS and 1% streptomycin, mixing equal amount of above mixed solution with mononuclear cell suspension, and placing in 37 deg.C water bath. A96-well plate precooled at-20 ℃ is taken, 2 mu L of the mononuclear cell agarose mixed solution is added into each well, and a glue drop with the diameter of about 2mm is required to be formed at the center of the bottom of each well. The gel-drop-spread 96-well plate was placed at 4 ℃ for 15 minutes. RPMI-1640 medium containing 10% FBS, 300. mu.g/mL B-HA +1ng/mL LPS + 10% FBS, 300. mu.g/mL B-HA + 300. mu.g/mL HA +1ng/mL LPS + 10% FBS, 1nmol/L fMLP + 10% FBS was prepared, and after the agarose gel drops in the wells had solidified, 100. mu.L of the above reagents were added to each well separately, 4 replicates in each group. And culturing the 96-well plate in a 37 ℃ constant temperature incubator for 3 hours, taking out, observing the migration result under an inverted microscope, photographing and recording, and calculating the cell migration area by using Image processing software Image j. Venous blood from 3 different volunteers per collection excluded individual differences and ensured that the experiment was reproducible.

(3) Effect of higher concentration hyaluronic acid fragment B-HA on removal of human freshly extracted neutrophils neutrophilic granulocyte according to the specification of human venous blood neutrophilic granulocyte isolation kit, obtaining neutrophilic granulocyte and mononuclear cell, re-suspending human neutrophilic granulocyte with 1 × RPMI-1640 culture medium, and adjusting cell density to 3 × 10-8/mL. Placing 0.8% agarose sterilized solution in 37 deg.C water bath, mixing equal amount of agarose solution with 2 × RPMI-1640 culture medium containing 20% FBS and 1% streptomycin, mixing equal amount of above mixed solution with neutral granulocyte suspension, and placing in 37 deg.C water bath. A96-well plate precooled at-20 ℃ is taken, 2 mu L of the neutrophilic granulocyte agarose mixed solution is added into each well, and a glue drop with the diameter of about 2mm is required to be formed at the center of the bottom of each well. The gel-drop-plated 96-well plate was placed at 4 ℃ for 15 minutes, RPMI-medium containing 10% FBS, 1. mu.g/mL Anti-CD44 Anti-body + 10% FBS, 300. mu.g/mL B-HA + 10% FBS, 300. mu.g/mL B-HA + 300. mu.g/mL HA + 10% FBS, 1ng/mL LPS + 10% FBS, 1nmol/L fMLP + 10% FBS was prepared, and after the in-well agarose gel drops had solidified, 100. mu.L of the above-mentioned reagent was added to each well, 4 replicates in each group. And culturing the 96-well plate in a 37 ℃ constant temperature incubator for 3 hours, taking out, observing the migration result under an inverted microscope, photographing and recording, and calculating the cell migration area by using Image processing software Image j. Venous blood from 3 different volunteers per collection excluded individual differences and ensured that the experiment was reproducible.

(4) Effect of higher concentration of hyaluronic acid fragment B-HA on LPS-induced human neutrophil removal: neutrophile granulocytes and mononuclear cells are obtained according to the specification of a human venous blood neutrophile granulocyte separation kit, the human neutrophile granulocytes are resuspended by using 1 XRPMI-1640 culture medium, the cell density is adjusted to 3X 10-8/mL, 0.8% agarose sterile solution is placed in a water bath at 37 ℃, equal amount of agarose solution is taken to be mixed with 2 XRPMI-1640 culture medium containing 20% FBS and 1% streptomycin, equal amount of the mixed solution is respectively taken to be mixed with the neutrophile granulocytes suspension, the mixed solution is placed in a water bath at 37 ℃, a 96-well plate pre-cooled at-20 ℃ is taken, 2 mu L of the neutrophile granulocyte agarose mixed solution is added into each well, and a colloid drop with the diameter of about 2mm is required to be formed in the center of the bottom of the well. The well-plated 96-well plate was placed at 4 ℃ for 15 minutes, RPMI-1640 medium containing 10% FBS, 300. mu.g/mL B-HA +1ng/mL LPS + 10% FBS, 300. mu.g/mL B-HA + 300. mu.g/mL HA +1ng/mL LPS + 10% FBS, 1nmol/L fMLP + 10% FBS was prepared, and after the agarose gel drops in the wells had solidified, 100. mu.l of the above-mentioned reagent was added to each well, 4 replicates in each group. And culturing the 96-well plate in a 37 ℃ constant temperature incubator for 3 hours, taking out, observing the migration result under an inverted microscope, photographing and recording, and calculating the cell migration area by using Image processing software Image j. Venous blood from 3 different volunteers per collection excluded individual differences and ensured that the experiment was reproducible.

3. Safety study of intravenous injection of hyaluronic acid fragment B-HA in beagle 30 healthy beagle dogs were each intravenously injected with 20mg/mL of sterile hyaluronic acid fragment B-HA injection (endotoxin <0.1EU/mL) at pH 6.5-7.5, and the experiment was repeated 7 days later, to observe whether beagle dogs were quiet, had pain-related avoidance of struggle and howling, had tired, trembled, convulsion, could not ride up, and died during and after injection.

4. Statistical analysis

Using a set of t-test comparisons, p >0.05(ns) was considered to have no statistical significance, p <0.05 (. sup.) -was considered to have statistical significance, and p <0.01 (. sup.) -or p <0.001 (. sup.) -was considered to have high statistical significance.

As a result:

(1) 99mTc labeling and tissue distribution of hyaluronic acid fragment B-HA study iQID dynamic imaging of mouse systemic tissue distribution after intravenous injection of 99mTc-B-HA is shown in fig. 6, and quantitative distribution in major organ tissues is shown in table 6. The results showed that 99mTc-B-HA rapidly distributed to the spleen, liver, and second lung 5 minutes after intravenous injection. The portion of 99mTc-B-HA is eliminated by the kidney and distributed to the bladder. Dynamic imaging demonstrated that 99mTc-B-HA is rapidly cleared in blood with a blood half-life of about 5 minutes. After 3 hours, only less than 20% of the initial 99mTc-B-HA remained in the circulating blood. The blood concentration of 99mTc-B-HA relative to other tissues is low, indicating that the tissue uptake of 99mTc-B-HA is very rapid.

TABLE 6 quantitative distribution of 99mTc-B-HA in major organ tissues after intravenous injection

(2) 125-I labeling and molecular imaging studies of hyaluronic acid fragment B-HA

¹²⁵ I-B-HA was injected subcutaneously into the lymph nodes and spleen of lymphoid organs within 5 minutes via the ends of the lower extremities of C57BL/6J mice. This result surprisingly shows that B-HA is specific to the lymphatic system after subcutaneous and intravenous injectionThe strong affinity suggests that the drug carries water and enters lymph reflux from inflammatory tissues together with human mononuclear cells. The effect of B-HA on lymphocyte reflux HAs certain effect on normalization of lymphocyte and related immunoregulation.

Discussion:

the present application for the first time found that 99mTc-B-HA had spread to lymph nodes and spleen of lymph organs after 5 minutes of subcutaneous and intravenous injection (FIGS. 6, 7; Table 6), suggesting that 99mTc-B-HA was rapidly absorbed by the hyaluronic acid receptor LYVE-1 in lymph nodes and spleen of lymph organs after intravenous injection [1,2,3,8,9,48,58,59 ]. The unpublished results of this study also support the effect of hyaluronic acid and hyaluronic acid fragments on rapid absorption through the lymphatic system and absorption and transport of interstitial fluid from inflammatory areas following subcutaneous injection as suggested by the relevant literature [48,58,59].

And (4) conclusion:

(1) the unpublished research result of the research also supports the function that the hyaluronic acid and the hyaluronic acid fragment are particularly quickly absorbed by a lymphatic system and absorbed and transported by tissue fluid in an inflammation area after being subcutaneously injected, which is shown by related documents; (2) the results herein show that labeled B-HA enters lymph nodes and spleen of lymphoid organs particularly rapidly after subcutaneous and intravenous injection, suggesting its involvement in lymphoid and monocyte immunoregulatory functions.

2. Effect of higher concentrations of hyaluronic acid fragments B-HA on the removal of human neutrophils and monocytes (mainly human lymphocytes and a few human monocytes) from lymph fluid

(1) Effect of higher concentrations of hyaluronic acid fragments B-HA on removal of freshly extracted mononuclear cells from humans

The results of FIG. 8 show that higher concentrations (300. mu.g/mL) of 35kDa hyaluronic acid B-HA and 1600kDa hyaluronic acid HA facilitate the removal of freshly extracted mononuclear cells (predominantly lymphocytes and a small number of macrophage precursor mononuclear cells), that mixed HA (300. mu.g/mL) of B-HA (300. mu.g/mL) further facilitates the removal of freshly extracted mononuclear cells, and that anti-CD44 antibodies, LPS and fMLP all facilitate the removal of freshly extracted mononuclear cells (FIG. 8), indicating a reliable process. The results suggest that injection of B-HA into the lymphatic system with high hyaluronic acid HA concentrations already present further promotes monocyte (mainly lymphocytes and a small number of macrophage precursor monocytes) removal and return to blood circulation.

(2) Effect of higher concentrations of hyaluronic acid fragment B-HA on LPS-induced human monocyte depletion

The study used freshly extracted monocytes (mainly lymphocytes and a small number of macrophage precursor monocytes), and both LPS and fMLP facilitated the removal of human freshly extracted monocytes (fig. 9), indicating that the study procedure is reliable. The results show that the higher concentration (300. mu.g/mL) of the 35kDa hyaluronic acid fragment B-HA did not significantly affect the LPS (1ng/mL) -induced removal of human freshly extracted mononuclear cells, the higher concentration (300. mu.g/mL) of the 1600kDa hyaluronic acid HA significantly promoted the LPS (1ng/mL) -induced removal of human freshly extracted mononuclear cells, and the higher concentration (300. mu.g/mL) of the B-HA mixed with the higher concentration (300. mu.g/mL) of HA significantly inhibited the LPS-induced removal of human freshly extracted mononuclear cells (FIG. 9).

(3) Effect of higher concentration of hyaluronic acid fragments B-HA on removal of human freshly extracted neutrophils

The results of this study show that the higher concentration (300. mu.g/mL) of 1600kDa hyaluronic acid HA and the 35kDa hyaluronic acid fragment B-HA normally (i.e. in the absence of endotoxin LPS) inhibit the removal of human freshly extracted neutrophils to essentially the same extent (FIG. 10), suggesting that HA and B-HA have the same affinity and biological effect on the hyaluronic acid receptor CD44 of neutrophils. Removal of human freshly extracted neutrophils was facilitated using anti-human CD44antibody (fig. 10). The result is combined with the result that dexamethasone inhibits the removal of the neutrophils (unpublished data), and the suggestion that the local part of a human body (particularly nerve tissues and lymph tissues with higher content of hyaluronic acid) inhibits the removal of the neutrophils by using the injection of the hyaluronic acid fragment B-HA with higher concentration and HAs anti-inflammatory effect.

(4) Effect of higher concentrations of hyaluronic acid fragment B-HA on LPS-induced human neutrophil migration

The results of this study show that the higher concentration (300. mu.g/mL) of 35kDa hyaluronic acid fragment B-HA, 1600kDa hyaluronic acid HA and mixed HA of B-HA significantly inhibited endotoxin LPS (1ng/mL) -induced removal of human freshly extracted neutrophils (FIG. 11), suggesting that HA and B-HA have similar affinity and biological effects on the hyaluronic acid receptors CD44 and Siglec-9 of neutrophils. LPS and fMLP facilitate the removal of freshly extracted human neutrophils, demonstrating the reliability of the present method.

In summary, the results of the above study, FIGS. 8 and 9 in conjunction with FIGS. 10 and 11, show that higher concentrations (300. mu.g/mL) of B-HA and HA in the presence and absence of LPS had different or opposite effects on the removal of freshly extracted monocytes and the removal of neutrophils.

Discussion:

the content of hyaluronic acid and hyaluronic acid fragments is at least 85-180 times higher than that of serum (0.01-0.1 mug/g tissue) in the breast lymph node and the thymus chylomicron [1], which indicates that the hyaluronic acid and hyaluronic acid fragments with higher concentration are concentrated by the lymphatic system and have effect on the lymphatic system. The relevant literature suggests that an adult has 10-100mg of hyaluronic acid and hyaluronic acid fragments per day entering the blood through the lymphatic system [1-3,7,46-47 ]. The results herein figures 8,9,10, 11 show that the higher concentrations (300. mu.g/mL) of hyaluronic acid fragment B-HA and hyaluronic acid HA in the presence and absence of endotoxin LPS have completely different effects on the removal of mononuclear cells and the removal of neutrophils freshly extracted from humans. It was first found herein that a higher concentration (300 μ g/mL) of the hyaluronic acid fragment B-HA promotes monocyte (mainly lymphocytes and a small number of macrophage precursor monocytes) dislodgement (homing), i.e. return to blood circulation within the lymphatic vessels (fig. 8,9) [4,5,6,9,10,48 ]. It is not clear herein whether these effects are mediated by the hyaluronic acid receptor LYVE-1 alone or in combination with CD44 [1,4,5,8,48 ]. The B-HA HAs the same water carrying function as HA, promotes lymphocyte reflux and promotes reabsorption of extracellular fluid in inflammatory tissues. The relevant literature supports the disappearance of mucocutaneous inflammation, clinically manifested as red swelling and hot pain, by the action of lymphatic reflux [1,4,5,6,8,9,10,14-19,48] it was found herein that higher concentrations (300 μ g/mL) of hyaluronic acid fragment B-HA, hyaluronic acid HA and B-HA mixed HA inhibit the removal of human neutrophils both in the presence and in the absence of endotoxin LPS (fig. 10, 11). Commercial products containing high concentrations of 1-2% (10-20mg/mL) hyaluronic acid fragment B-HA have been previously reported by the authors in local journals and patent applications to have utility in the treatment of topical human skin and of the inflammatory, red, hot pain of the human pharyngeal mucositis [14-19 ]. Therefore, the above results support the effect of topical application of high concentrations of hyaluronic acid fragments B-HA in the treatment of inflammatory red swelling and pain of the skin mucosa and further support the physiological phenomenon that neutrophil entry is rarely found in the lymphatic system with high concentrations of hyaluronic acid (fig. 10, 11). In addition, the related literature indicates that the content of hyaluronic acid and hyaluronic acid fragments in central nervous tissue is at least 330-fold 1150-fold higher than that in serum (0.01-0.1. mu.g/g tissue), suggesting that the hyaluronic acid fragment B-HA with higher concentration of 35kDa also HAs a role in brain tissue [1 ].

The hyaluronic acid fragments B-HA with good permeability interact with various cell receptors of human body after being injected, including LYVE-1 (macrophage, DC cell, T cell, B cell), CD44 (erythrocyte, leukocyte and bone marrow cell), RHAMM (leukocyte, microglia, endothelial cell and muscle cell), Siglec-9 (neutrophil, monocyte, DC cell), TLR2 (macrophage, DC cell, T cell, B cell, monocyte, microglia) and HARE (liver, lymph node and spleen sinus endothelial cell), CEMIP (fibroblast, epithelial cell and various tumor cells), and TMEM2 (various tumor cells), and participate in the regulation of various functions in vivo [1,4-6,8-30], and also play important functions in the body of non-wonderful animal naked mole [31-37 ]. The research results and related documents show that the clinical effect of the hyaluronic acid fragment B-HA injection on human bodies can only be a comprehensive result of the combination of different cells and different tissues and organs with various receptors, and further large-dose animal experiments or direct human body comprehensive clinical effect research is supported.

And (4) conclusion:

1. the results of the study herein (FIGS. 8,9,10, 11) show that higher concentrations (300. mu.g/mL) of B-HA and HA do not have the same or opposite effects on monocyte and neutrophil removals from fresh human extracts in the presence and absence of LPS; 2. the results herein show that a higher concentration (300 μ g/mL) of the 35kDa hyaluronic acid fragment B-HA facilitates the removal of freshly extracted human monocytes (with lymphocytes as the primary and the minority of human monocytes), suggesting that the hyaluronic acid fragment B-HA and the water it carries reflux with human monocytes from the inflammatory tissue into the lymphatic system; 3. the results further show that the higher concentration of the hyaluronic acid fragment B-HA can obviously inhibit the removal of neutrophils in the presence and absence of endotoxin LPS, and the local tissues have anti-inflammatory effect by using the higher concentration of the hyaluronic acid fragment B-HA; 4. the research results and literature researches indicate that the clinical effect of the hyaluronic acid fragment B-HA injection of 35kDa on human bodies can only be a comprehensive result of the combination of different cells and different tissues and organs with various receptors, and further research on the comprehensive clinical effect of the hyaluronic acid fragment B-HA injection on human bodies is supported.

3. Safety study of intravenous injection of hyaluronic acid fragment B-HA in beagle dogs 30 beagle dogs were intravenously injected with 20mg/mL of sterile B-HA injection solution (endotoxin <0.1EU/mL) at pH 6.5-7.5 per injection, and no local pain and vascular occlusion of the injection and physical symptoms due to allergic reaction were observed.

TABLE 7 safety study of beagle dogs intravenously injected with hyaluronic acid fragment B-HA

Discussion:

the beagle dogs injected with 100mg of B-HA injection each time had an average body weight of 15 kg, about 1000 mL of blood, and the concentration of B-HA in the blood was up to l00 μ g/mL, although significant red blood cell aggregation and blood sedimentation increase were induced by more than 0.15% (1500 μ g/mL) and more than 0.08% (800 μ g/mL) of the hyaluronic acid fragment B-HA, but the beagle dogs struggled, eluded and died during the experiment without local pain and blood vessel occlusion by injection and allergic reaction (Table 7). The hyaluronic acid fragment B-HA injection liquid provided by the invention is designed to be injected into a person with the average weight of 70 kilograms at 100mg subcutaneous deep fat or a diseased pain place every time, the person with the average weight of 70 kilograms HAs about 7 liters of blood, and the hyaluronic acid fragment B-HA concentration in the blood is up to 14.2 mug/mL (0.0014%) and is not enough to cause obvious erythrocyte aggregation and erythrocyte sedimentation increase under the assumption that the 100mg subcutaneous deep fat injection of the hyaluronic acid fragment B-HA injection liquid is completely absorbed in 7 liters of blood at once. Therefore, the hyaluronic acid fragment B-HA is safe to use by injection.

And (4) conclusion:

the result shows that the safety of the hyaluronic acid fragment B-HA injection with the average molecular weight of 35kDa for intravenous injection of beagle dogs is good.

Example 3

The purpose is to use commercial bovine extracted hyaluronidase PH20 injection and high molecular hyaluronic acid injection to prepare B-HA or HA35 with average molecular weight of 35.4kDa in a short time for treating hypersensitive inflammatory reaction of local large-area red swelling and hard pain caused by mosquito bite.

The method comprises the following steps:

12 cases of patients with hypersensitive reaction (rapidly-generated large-area inflammation and hard pain at local part of bite) of mosquito (mosquito or bumblebee). The following two injections were mixed at 37 degrees for 20 minutes and injected into the affected area, and then the change of large area red swelling and pain on the area of bite after treatment was observed.

Before injection, bovine testis hyaluronidase PH20 (1500U/count, H31022111, Shanghai first chemical pharmaceutical factory) and sodium hyaluronate (hyaluronic acid) injection (trade name of Schopper) with an average molecular weight of 1600kDa (20mg/2ml) are mixed according to the ratio of hyaluronidase 20000U/g hyaluronic acid, and are subjected to enzymolysis at 37 deg.C for 20 minutes in a sufficient amount or in a slight excess amount to produce B-HA or HA35 with an average molecular weight of 35.4kDa (refer to example 1 and figure 3). The prepared B-HA or HA35 with the average molecular weight of 35.4kDa is used for injection at the red and hard part of mosquito or bee bites after skin test. Note: the mixed solution prepared in FIG. 3 of example 1 above was used to leave a sample and the average molecular weight was measured to be 35.4kDa by gel electrophoresis and 18-angle laser.

As a result:

the commercial hyaluronidase PH20 injection and the polymer hyaluronic acid injection are used for preparing the B-HA with the average molecular weight of 35.4kDa or the high-sensitivity inflammatory reaction of the large-area local red swelling and hard pain caused by mosquito bite by local injection at the affected part of HA35 in a short time.

Table 8, hyaluronic acid fragment B-HA with an average molecular weight of 35.4kDa or HA35 is prepared by using commercial hyaluronidase PH20 injection and high-molecular hyaluronic acid injection in a short time, and is locally injected to treat the condition of hypersensitive inflammatory response of local large-area red swelling and pain caused by mosquito bite.

Discussion:

this rapid therapeutic effect may be associated with the effect of hyaluronic acid fragment B-HA in promoting inflammatory edema lymphatic fluid and lymphocyte reflux and local anti-inflammatory effects.

And (4) conclusion:

a method for preparing hyaluronic acid fragment for rapidly treating hypersensitive inflammatory reaction of local large-area red swelling and hard pain caused by mosquito (mosquito or bumblebee) bite by using commercial hyaluronidase PH20 injection extracted from bovine testis and directly mixing commercial high-molecular hyaluronic acid injection and an application in preparing a medicament.

Reference documents:

1 Jackson D G.Hyaluronan in the lymphatics:The key role of the hyaluronan receptor LYVE-1 in leucocyte trafficking.Matrix Biol,2019,78-79:219-235.

2 McDonald B,Kubes P.Interactions between CD44 and Hyaluronan in Leukocyte Trafficking.Front Immunol,2015,6:68.

3 Jackson D G.Leucocyte Trafficking via the Lymphatic Vasculature-Mechanisms and Consequences.Front Immunol,2019,10:471.

4 Fraser J R E,Laurent T C,Laurent U B G.Hyaluronan:its nature,distribution,functions and turnover.Journal InternalMedicine,1997,242(1):27-33.

5 Lena,Lebel.Clearance of hyaluronan from the circulation.Advanced Drug Delivery Reviews,1991,7:221-235.

6 Laurent U B G,Reed R K.Turnover of hyaluronan inthe tissues.Advanced Drug Delivery Reviews,1991,7(2):237-256.

7 Laurent U B G,Laurent T C.On the origin of hyaluronan in blood.Biochemistry International,1981,2(2):195-199.

8 Tengblad A,Laurent U B G,Lilja K,et al.Concentration and relative molecular mass of hyaluronate in lymph and blood.Biochemical Journal,1986,236(2):521-525.

9 Fraser J R,Kimpton W G,Laurent T C,et al.Uptake and degradation of hyaluronan in lymphatic tissue.Biochemical Journal,1988,256(1):153.

10 Reed R K,Laurent U B,Fraser J R,et al.Removal rate of[3H]hyaluronan injected subcutaneously in rabbits.American Journal of Physiology Heart&Circulatory Physiology,1990,259(2):H532-H535.

11 Ji R R,Chamessian A,Zhang Y Q.Pain regulation by non-neuronal cells and inflammation.Science,2016,354(6312):572-577.

12 Chen G,Zhang Y Q,Qadri Y J,et al.Microglia in Pain:Detrimental and Protective Roles in Pathogenesis and Resolution of Pain.Neuron,2018,100(6):1292-1311.

13 Haight E S,Forman T E,Cordonnier S A,et al.Microglial Modulation as a Target for Chronic Pain:From the Bench to the Bedside and Back.Anesth Analg,2019,128(4):737-746.

14 Wang M J,Kuo J S,Lee W W,et al.Translational event mediates differential production of tumor necrosis factor-alpha in hyaluronan-stimulated microglia and macrophages.J Neurochem,2006,97(3):857-71.

15 Austin J W,Gilchrist C,Fehlings M G.High molecular weight hyaluronan reduces lipopolysaccharide mediated microglial activation.J Neurochem,2012,122(2):344-55.

16 Jensen G,Holloway J L,Stabenfeldt S E.Hyaluronic Acid Biomaterials for Central Nervous System Regenerative Medicine.Cells,2020,9(9):2113.

17 Shen M Q,Liu X,Wei C,et al.Clinical study of 35kDa hyaluronan fragment B-HA on laser-induced inflammatory skin wound.Progress in Modern Biomedicine,2015,15(7):1300-1304.

18 Hui M Z.Expert opinion:clinical effect of inflammation control by frozen cold pack,ozonated water and hyaluronan fragment on wound healing.E-Journal of General Surgery,2015,3(4):8-11.

19 Zhao H D,Hui M Z.Preliminary study of 35kDa hyaluronan fragment B-HA on chronic pharyngitis.Clinical Journal of Medical Officers,2014,42(8):864-867.

20 Zhao H D,Hui M Z.Preliminary study of 35kDa hyaluronan fragment B-HA on laryngopharyngeal reflux disease.E-Journal of Clinical Medical Literature,2016,3(24):4898-4899.

21 Zhang H W,Wei W R,Cheng X F,et al.Clinical study of 35kDa hyaluronan fragment B-HA tooth brush gel on gingivitis.E-Journal of Clinical Medical Literature,2015,2(23):4808-4809.

22 Huang P F,Feng P,Shuang B,et al.Clinical study of 35kDa hyaluronan fragment B-HA tooth brush gel on puberty gingivitis.Medical Journal of Air Force,2020,36(1):71-74.

23 Chanmee T,Ontong P,Itano N.Hyaluronan:A modulator of the tumor microenvironment.Cancer Lett,2016,375(1):20-30.

24 Turley E A,Wood D K,McCarthy J B.Carcinoma Cell Hyaluronan as a"Portable"Cancerized Prometastatic Microenvironment.Cancer Res,2016,76(9):2507-12.

25 Kuang D M,Wu Y,Chen N,et al.Tumor-derived hyaluronan induces formation of immunosuppressive macrophages through transient early activation of monocytes.Blood,2007,110(2):587-95.

26 Zhang G,Guo L,Yang C,et al.A novel role of breast cancer-derived hyaluronan on inducement of M2-like tumor-associated macrophages formation.Oncoimmunology,2016,5(6):e1172154.

27 Kobayashi N,Miyoshi S,Mikami T,et al.Hyaluronan deficiency in tumor stroma impairs macrophage trafficking and tumor neovascularization.Cancer Res,2010,70(18):7073-83.

28 Lim H Y,Lim S Y,Tan C K,et al.Hyaluronan Receptor LYVE-1-Expressing Macrophages Maintain Arterial Tone through Hyaluronan-Mediated Regulation of Smooth Muscle Cell Collagen.Immunity,2018,49(2):326-341.

29 Wang N,Liu C,Wang X,et al.Hyaluronic Acid Oligosaccharides Improve Myocardial Function Reconstruction and Angiogenesis against Myocardial Infarction by Regulation of Macrophages.Theranostics,2019,9(7):1980-1992.

30 Kerfoot S M,McRae K,Lam F,et al.A novel mechanism of erythrocyte capture from circulation in humans.Exp Hematol,2008,36(2):111-8.

31 Secundino I,Lizcano A,Roupé K M,et al.Host and pathogen hyaluronan signal through human siglec-9 to suppress neutrophil activation.J Mol Med(Berl),2016,94(2):219-33.

32 Lizcano A,Secundino I,

et al.Erythrocyte sialoglycoproteins engage Siglec-9 on neutrophils to suppress activation.Blood,2017,129(23):3100-3110.

33 Kiser Z M,Lizcano A,Nguyen J,et al.Decreased erythrocyte binding of Siglec-9 increases neutrophil activation in sickle cell disease.Blood Cells Mol Dis,2020,81:102399.

34 Smith E S,

Lechner S G,et al.The molecular basis of acid insensitivity in the African naked mole-rat.Science,2011,334(6062):1557-60.

35 Kulaberoglu Y,Bhushan B,Hadi F,et al.The material properties of naked mole-rat hyaluronan.Sci Rep,2019,9(1):6632.

36 Seluanov A,Gladyshev V N,Vijg J,et al.Mechanisms of cancer resistance in long-lived mammals.Nat Rev Cancer,2018,18(7):433-441.

37 Tian X,Azpurua J,Hine C,et al.High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat.Nature,2013,499(7458):346-9.

38 Takasugi M,Firsanov D,Tombline G,et al.Naked mole-rat very-high-molecular-mass hyaluronan exhibits superior cytoprotective properties.Nat Commun,2020,11(1):2376.

39 Cowman M K,Lee H G,Schwertfeger K L,et al.The Content and Size of Hyaluronan in Biological Fluids and Tissues.Front Immunol,2015,6:261.

40 Kobayashi T,Chanmee T,Itano N.Hyaluronan:Metabolism and Function.Biomolecules,2020,10(11):1525.

41 Chowdhury B,Hemming R,Hombach-Klonisch S,et al.Murine hyaluronidase 2 deficiency results in extracellular hyaluronan accumulation and severe cardiopulmonary dysfunction.J Biol Chem,2013,288(1):520-8.

42 Chowdhury B,Xiang B,Muggenthaler M,et al.Hyaluronidase 2 deficiency is a molecular cause of cor triatriatum sinister in mice.Int J Cardiol,2016,209:281-3.

43 Chowdhury B,Xiang B,Liu M,et al.Hyaluronidase 2 Deficiency Causes Increased Mesenchymal Cells,Congenital Heart Defects,and Heart Failure.Circ Cardiovasc Genet,2017,10(1):e001598.

44 Matsumoto K,Li Y,Jakuba C,et al.Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth,patterning,chondrocyte maturation and joint formation in the developing limb.Development,2009,136(16):2825-35.

45 Huang Y,Askew E B,Knudson C B,et a l.CR I SPR/Cas9 knockout of HAS2 i n rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention.Matrix Biol,2016,56:74-94.

46 Shimoda M,Yoshida H,Mizuno S,et al.Hyaluronan-Binding Protein Involved in Hyaluronan Depolymerization Controls Endochondral Ossification through Hyaluronan Metabolism.Am J Pathol,2017,187(5):1162-1176.

47 Homann S,Grandoch M,Kiene L S,et al.Hyaluronan synthase 3 promotes plaque inflammation and atheroprogression.Matrix Biol,2018,66:67-80.

48 Bahrami S B,Tolg C,Peart T,et al.Receptor for hyaluronan mediated motility(RHAMM/HMMR)is a novel target for promoting subcutaneous adipogenesis.Integr Biol(Camb),2017,9(3):223-237.

49 Rishton G,HUI M Z.A“GC-rich”method for mammalian gene expression:A dominant role of non-coding DNA GC content in the regulation of mammalian gene expression.Science China(Life Sciences),2010,53(01):94-100.

50 Hunnicutt G R,Primakoff P,Myles D G.Sperm surface protein PH-20 is bifunctional:one activity is a hyaluronidase and a second,distinct activity is required in secondary sperm-zona binding,Biology of Reproduction,1996,55:80-86.

51 Locke K W,Maneval D C,LaBarre M J.

drug delivery technology:a novel approach to subcutaneous administration using recombinant human hyaluronidase PH20.Drug Deliv,2019,26(1):98-106.

52 Beech D J,Madan A K,Deng N.Expression of PH-20 in Normal and Neoplastic Breast Tissue.Journal of Surgical Research,2002,103:203–207.

53 David R Hill,Sean P Kessler,Hyunjin K Rho,et al.Specific-sized hyaluronan fragments promote expression of human beta-defensin 2 in intestinal epithelium.The Journal of Biological Chemistry,2013,287(36):30610-30624.

54 Hil D Rl,Rho H K,Kessler S P,et al.Human milk hyaluronan enhances innate defense of the intestinal epithelium.The Journal of Biological Chemistry,2013,288(40):29090-2914.

55 Kessler S P,Obery D R,Nickerson K P,et al.Multifunctional role of 35 kilodalton hyaluronan in promoting defense of the intestinal epithelium.Journal of Histochemistry&Cytochemistry,2018,66(4):273-287.

56 Kim Y,Kessler S P,OberyD R,et al.Hyaluronan 35kDa treatment protects mice from citrobacter rodentium in fection and induces epithelial tight junctional protein ZO-1 in vivo.Matrix Biology,2017,62:28-39.

57 Gunasekaran A,Eckert J,Burge K,et al.Hyaluronan 35kDa enhances epithelial barrier function and protects against the development of murine necrotizing enterocolitis.Pediatric Research,2019,87(7):1177-1184.

58 Laznicek M,Laznickova A,Cozikova D,Velebny V:Preclinical pharmacokinetics of radiolabelled hyaluronan.Pharmacol Rep 2012,64(2):428-437.

59 Huang C,Chen F,Zhang L,Yang Y,Yang X,Pan W:(99m)Tc Radiolabeled HA/TPGS-Based Curcumin-Loaded Nanoparticle for Breast Cancer Synergistic Theranostics:Design,in vitro and in vivo Evaluation.Int J Nanomedicine 2020,15:2987-2998.

60 Luquita A,Urli L,Svetaz M J,et al.In vitro and ex vivo effect of hyaluronic acid on erythrocyte flow properties.J Biomed Sci,2010,17(1):8.

61 Melder R J,Yuan J,Munn L L,et al.Erythrocytes enhance lymphocyte rol l ing and arrest in vivo.Microvasc Res,2000,59(2):316-22.

62 Ke Z H.Stem Cell Maintenance in Naked Mole Rats and other Longevity Mechanisms in Rodents”.PhD Thesis,University of Rochester Rochester,2018.

63

Mendichi R,KoganG,et al.Degradative Action of Reactive Oxygen Species on Hyaluronan.Biomacromolecules,2006,7(3):659-668.

64Wright H L,Moots R J,Bucknall R C,et al.Neutrophil function in inflammation and inflammatory diseases.Rheumatology(Oxford),2010,49(9):1618-31.

the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention in any way, and it will be apparent to those skilled in the art that the above description of the present invention can be applied to various modifications, equivalent variations or modifications without departing from the spirit and scope of the present invention.

Claims

1. A novel use of a hyaluronic acid fragment, wherein the hyaluronic acid fragment is: the hyaluronic acid fragment which is prepared by using recombinant human hyaluronidase PH20 or extracted bovine hyaluronidase PH20 and sufficiently performing enzymolysis on high or medium molecular weight hyaluronic acid raw materials in a sufficient or slight excess manner, can pass through a filter membrane with the pore diameter of 0.22 mu m and has the average molecular weight of 35 +/-8 KDa;

the application is as follows:

use of the hyaluronic acid fragments as a human monocyte migration-promoting agent;

and/or, the use of said fragments of hyaluronic acid as immunomodulators of human lymphocytes and fluid reflux and lymphocytes by subcutaneous or intravenous injection;

and/or the use of said hyaluronic acid fragments as inhibitors of human neutrophil removal.

2. A novel use of a hyaluronic acid fragment, wherein the hyaluronic acid fragment is: hyaluronic acid fragments which are prepared by using recombinant human hyaluronidase PH20 or extracted bovine hyaluronidase PH20, sufficiently or slightly excessively and sufficiently performing enzymolysis on high or medium molecular weight hyaluronic acid raw materials, can pass through a filter membrane with the pore diameter of 0.22 mu m, and have the average molecular weight of 35 +/-8 KDa;

the application is as follows:

the application of the hyaluronic acid fragment in preparing a medicament for treating inflammatory diseases related to human monocyte removal or preparing a human monocyte removal promoter;

and/or the use of the hyaluronic acid fragments for the preparation of a medicament for the treatment of inflammatory diseases associated with reflux of human lymphocytes and fluid, by subcutaneous or intravenous injection, or for the preparation of an immunomodulator for human lymphocytes and fluid reflux and lymphocytes by subcutaneous or intravenous injection;

and/or, the application of the hyaluronic acid fragment in preparing a medicament for treating inflammatory diseases related to human neutrophil removal, or preparing an inhibitor for treating human neutrophil removal.

3. The novel use of the hyaluronic acid fragments of claim 1 or 2, wherein the human mononuclear cells are lymphocytes;

the hyaluronic acid fragment inhibited human neutrophil removal at a concentration of 150-300. mu.g/mL.

4. A method for producing a hyaluronic acid fragment, characterized by using recombinant human hyaluronidase PH20 or extracted bovine hyaluronidase PH20, and sufficiently or slightly excessively digesting a high or medium molecular weight hyaluronic acid material to produce a hyaluronic acid fragment having an average molecular weight of 35 + -8 KDa.

5. The method for producing the hyaluronic acid fragment of claim 4, wherein the fragment can pass through a 0.22 μm-pore filter; the enzymolysis time is 2-6 hours.

6. The method for producing the hyaluronic acid fragments according to claim 4, wherein the hyaluronic acid fragment injection having an average molecular weight of 35 ± 8kDa is prepared by mixing a high or medium molecular weight hyaluronic acid injection with the recombinant human hyaluronidase PH20 or the extracted bovine hyaluronidase PH20 injection, and subjecting the mixture to enzymatic hydrolysis.

7. The method for producing the hyaluronic acid fragment according to claim 4 or 6, wherein the enzymolysis time is 10 to 20 minutes.

8. The novel use according to any one of claims 1 to 3, or the method of manufacture according to any one of claims 4 to 7, wherein the sufficient or slight excess is sufficient enzymatic hydrolysis to:

(1) the molecular weight of the hyaluronic acid fragments prepared by sufficiently or slightly excessively performing enzymolysis on the neutralized high molecular weight hyaluronic acid raw material for 10-20 minutes is basically consistent with that of the hyaluronic acid fragments prepared by sufficiently or slightly excessively performing enzymolysis on the neutralized high molecular weight hyaluronic acid raw material for 1-6 hours, and the coefficient of variation CV is less than 15%;

(2) the low molecular hyaluronic acid fragment product of which more than 99 percent of high or medium molecular hyaluronic acid raw material is sufficiently enzymolyzed by enough or slight excess, is completely and smoothly filtered by a 0.22um pore size filter membrane;

(3) the hyaluronidase activity has no residue or little residue after sufficient or slight excess and sufficient enzymolysis reaction, the residue is less than 15 percent, and the hyaluronidase can be completely inactivated in 45 minutes at 80 ℃.

9. A method for producing hyaluronic acid fragments having different molecular weights, comprising: recombinant hyaluronidases with different molecular weights or extracted hyaluronidases are used for carrying out enzymolysis and cutting on high molecular weight hyaluronic acid raw materials to prepare hyaluronic acid fragments with different molecular weights.

10. The method for producing the hyaluronic acid fragment according to claim 9, wherein the recombinant hyaluronidase or extracted hyaluronidase having a different molecular weight is: full-length, partial and fused recombinant hyaluronidases or extracted hyaluronidases of a variety of different species.